Alpha-amylase variants with altered properties

ABSTRACT

Disclosed are compositions comprising variants of alpha-amylase that have alpha-amylase activity and which exhibit altered properties relative to a parent AmyS-like alpha-amylase from which they are derived. The compositions comprise an additional enzyme such as a phytase. Also disclosed are methods of using the compositions, and kits related thereto.

CROSS-REFERENCE TO RELATED APPLICATIONS

This claims benefit of U.S. Provisional Applications 60/985,619, filedNov. 5, 2007, 61/026,579, filed Feb. 6, 2008, 61/041,075, filed Mar. 31,2008, and 61/059,411, filed Jun. 6, 2008, the disclosures of each ofwhich are incorporated herein by reference in their entireties, for allpurposes.

SEQUENCE LISTING

Attached hereto is a sequence listing comprising SEQ ID NOS 1-31, eachof which is herein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

This disclosure relates to novel alpha-amylases. In particular, itrelates to certain alpha-amylase variant activities, as well as blendsthereof with one or more other enzymes, such as phytases.

BACKGROUND

Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1)constitute a group of enzymes that catalyze hydrolysis of starch andrelated linear or branched 1,4-glucosidic oligo- and polysaccharides.

Alpha-amylases can be used for a variety of purposes. For examples,alpha-amylases are used commercially in the initial stages of starchprocessing (e.g., liquefaction); in wet milling processes; and inalcohol production from carbohydrate sources. They are also used ascleaning agents or adjuncts in detergent matrices; in the textileindustry for starch desizing; in baking applications; in the beverageindustry; in oilfields in drilling processes; in recycling processes,e.g. for de-inking paper, and in animal feed.

Attempts have been made to construct alpha-amylase variants withimproved properties for specific uses, such as starch liquefaction andtextile desizing.

There is a need for the creation and improvement of amylases thatprovide, e.g., manufacturing and/or performance advantages over theindustry standard enzymes (e.g., from Bacillus licheniformis), forvarious uses, including commercial processing of grain, e.g.,liquefaction processes. There is also a need for compositions comprisingimproved amylases and additional enzymes, such as phytases.

SUMMARY

In one aspect, the present disclosure relates, inter alia, to novelα-amylolytic enzymes variants of parent α-amylase such as a AmyS-likeα-amylase, in particular variants exhibiting altered properties whichare advantageous in connection with the industrial processing of starch(starch liquefaction, saccharification and the like).

For example, the variant is altered, as compared to a parent AmyS-likealpha-amylase or a reference alpha-amylase, in one or more of netcharge, substrate specificity, substrate cleavage, substrate binding,thermal stability, activity at one or more pH's, stability at one ormore pH's [such as increased stability at particular pHs (e.g. low (e.g.pH<6, in particular, pH<5) or high (e.g. pH>9) pH values], stability inoxidizing conditions, metal ion requirements [for example, Ca²⁺dependency, or Ca²⁺ requirements], specific activity, catalytic rate,catalytic efficiency, activity in the presence of phytic acid or anotherphytates (i.e, susceptibility to inhibition by phytates), thermal or pHstability in the presence of phytic acid or a phytate, ability to effectpeak viscosity in a liquefaction test, or ability to effect finalviscosity in a liquefaction test, and other properties of interest. Forinstance, one or more alterations may result in a variant that hasreduced Ca²⁺ dependency and/or an altered pH/activity profile and/oraltered thermostability, as compared to a parent α-amylase, such as anAmyS-like alpha-amylase.

In one of its aspects, the disclosure relates to variant alpha-amylasescomprising an amino acid sequence at least 95% identical to that of aparent AmyS-like alpha-amylase, and having a substitution at an aminoacid position corresponding to position 242 of a referencealpha-amylase, and further comprising one or more of the followingmodifications to it amino acid sequence:

a) one or more of substitution at positions as follows: a cysteine atamino acid position 349, a cysteine at 428, a glutamic acid at 97, anarginine at 97, a glutamic acid at 319, an arginine at 319, a glutamicacid at 358, an arginine at 358, a glutamic acid at 443, or an arginineat 443;

b) other sequence modification at one or more amino acid positionscorresponding to amino acid positions 97, 319, 349, 358, 428, or 443;

c) deletion of one or more amino acids at positions F178, R179, G180,I181, G182, or K183, or pairs thereof;

d) other sequence modifications at one or more amino acid positions 178,179, 180, 181, 182, or 183;

e) substitution of N193F or V416G, or both;

f) other sequence modification at position 193, 416 or both;

g) substitution of one or more proline residues present in the part ofthe alpha-amylase variant that is modified, with an alanine, glycine,serine, threonine, valine or leucine residue.

h) substitution of one or more proline residues present in the part ofthe alpha-amylase variant that is modified, with anothernaturally-occurring amino acid residue;

i) substitution of one or more cysteine residues present in the parentalpha-amylase with a serine, alanine, threonine, glycine, valine orleucine residue;

j) substitution of one or more cysteine residues present in the parentalpha-amylase variant, with another naturally-occurring amino acidresidue;

k) where SEQ ID NO: 7 is the reference amylase for numbering, any of thefollowing mutations M15T,L, M15X, V128E, V128X, H133Y, H133X, N188S,T,P,N188X, M197T,L, M197X, A209V, A209X, M197T/W138F, M197T/138Y,M15T/H133Y/N188S, M15N128E/H133Y/N188S, E119C/S130C, D124C/R127C,H133Y/T149I, G475R, H133Y/S187D or H133Y/A209V.

l) other modification at one or more of positions M15, V128, A111, H133,W138, T149, M197, N188, A209, A210, H405, T412, where SEQ ID NO: 7 isthe reference amylase;

m) where the parent alpha-amylase comprises SEQ ID NO: 7, deletion orsubstitution of one or more of a cysteine residue (C363) or one or moremethionine residues located in any of positions M8, M9, M96, M200, M206,M284, M307, M311, M316 and M438 when SEQ ID NO: 2 is the referenceamylase;

n) modification of one or more amino acid residues corresponding to P17,D19, T21, N28, S51, G72, V74, A82, Q86, Q89, A93, G95, Q97, W115, D117,P123, S124, D125, N127, I130, G132, Q135, P145, G146, G148, S153, Y159,W166, S169, K171, R179, G180, I181, G182, K183, W187, P209, N224, S242,P245, G256, D269, N271, T278, N281, G302, A304, R308, T321, Q358, P378,S382, K383, T398, H405, T417, E418, P420, G421, P432, W437, G446, G454,S457, T459, T461, S464, G474, or R483, where SEQ ID NO: 1 or 2 are thereference amylase; or

o) a set of substitutions of a) Q97E, Q319E, Q358E, Q443E; b) Q97E,Q319R, Q358E, Q443R; c) Q97E, Q319R, Q358E; d) Q97E, Q319R, Q443E; e)Q97E, Q319R, Q443R; f) Q97E, Q358R; g) Q897E, Q443E; h) Q319R, Q358E,Q443E; or i) Q319R, Q358R, Q443E.

In presently preferred embodiments, the alpha-amylase variant is aS242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, orS242T variant.

The alpha-amylase variant is preferably derived from a parent AmyS-likealpha-amylase comprising any of SEQ ID NOs: 1, 2, 6, 7, 8, 9, 10, 11,12, 15, or 16. The reference alpha-amylase used for numbering the aminoacid residues is preferably SEQ ID NO: 1 or 2 in certain embodiments.

In one embodiment, the amino acid sequence of the variant is at least98% identical to that of a parent AmyS-like alpha-amylase.

Also provided are nucleic acids, i.e., polynucleotides, comprising anencoding sequence that encodes a) an amino acid sequence of the variantsdescribed herein; or b) any of SEQ ID NOs: 3, 4, 16, 22, 23, 24, 25, 26,27, 28, 29, or 30. Provided as well are vectors and host cellscomprising the polynucleotides or vectors.

In various embodiments, the host cell is a Bacillus subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, B. thuringiensis, Streptomyces lividans, S. murinus; Escherichiacoli, or a Pseudomonas spp.

In another aspect, there is provided herein a variant of a parentGeobacillus stearothermophilus alpha-amylase, wherein the variant has anamino acid sequence which has at least 95% homology to a parentGeobacillus stearothermophilus alpha-amylase and comprises asubstitution of amino acid 242, wherein the amino acid positions in thepeptide sequence are numbered relative to SEQ ID NO: 1 or 2, and whereinthe variant has alpha-amylase activity.

In another aspect, provided are compositions comprising:

-   -   a) an alpha-amylase variant as provided herein, and    -   b) at least one additional enzyme.

In one embodiment, the composition is one comprising a) at least onevariant amylase comprising an amino acid sequence at least 95% identicalto that of a parent AmyS-like alpha-amylase, and having a substitutionat an amino acid position corresponding to position 242 of a referencealpha-amylase, said variant having detectable alpha-amylase activity,and b) at least one additional enzyme.

The additional enzyme is preferably a phytase. The alpha-amylase variantin the composition is preferably a S242A, S242D, S242E, S242F, S242G,S242H, S242L, S242M, S242N, S242Q, or S242T variant.

In one embodiment, the alpha-amylase variant further comprises asequence modification at one or more amino acid positions correspondingto amino acid positions 97, 179, 180, 193, 319, 349, 358, 416, 428, or443 of the reference alpha-amylase. The alpha-amylase variant can alsocomprise one or more of substitution at positions as follows: a cysteineat 349, a cysteine at 428, a glutamic acid at 97, an arginine at 97, aglutamic acid at 319, an arginine at 319, a glutamic acid at 358, anarginine at 358, a glutamic acid at 443, or an arginine at 443.

In one embodiment, the alpha-amylase variant comprises a substitution ofan N193 or a V416 or both, e.g., a substitution of N193F or V416G, orboth.

The alpha-amylase variant comprises deletion of amino acids 179 and 180in other embodiments. The composition in one embodiment comprises analpha-amylase variant has at least 95% homology to SEQ ID NO: 2 andcomprises a substitution of amino acid 242 relative to numbering in areference alpha-amylase comprising the amino acid sequence SEQ ID NO: 1.As above, the parent AmyS-like alpha-amylase is preferably SEQ ID NO: 1,2, 6, 7, 8, 9, 10, 11, 12, 15, or 16.

In one aspect, the disclosure relates to hydrolyzing a soluble starchsubstrate using alpha-amylase (AA) activity and a phytate-hydrolyzingenzyme (FTU). For example, wherein the ratio of AAU:FTU is from about1:15 to about 15:1, preferably from 1:10 to about 10:1. In an embodimentthe ratio of AAU:FTU is from 1:4 to 3:1. In a further embodiment theratio of AAU:FTU is 1:1. In one presently preferred composition, thephytase has a sequence that is SEQ ID NO: 31.

More particularly, methods are provided for treating a starch slurrycomprising the steps of a) adding to the starch slurry at least onephytase and at least one alpha-amylase; wherein the phytase and thealpha-amylase are added at or about the same time, or separately in anyorder, and wherein the alpha-amylase is a variant amylase comprising anamino acid sequence at least 95% identical to that of a parent AmyS-likealpha-amylase, and having a substitution at an amino acid positioncorresponding to position 242 of a reference alpha-amylase, said varianthaving detectable alpha-amylase activity; and b) incubating the starchslurry under conditions permissive of activity of the phytase and thealpha-amylase.

The alpha-amylase variant for use in the method is preferably a S242A,S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242Tvariant. In one embodiment, the alpha-amylase variant further comprisesa sequence modification at one or more amino acid positionscorresponding to amino acid positions 97, 179, 180, 193, 319, 349, 358,416, 428, or 443 of the reference alpha-amylase. The alpha-amylasevariant can comprise one or more of substitution at positions asfollows: a cysteine at 349, a cysteine at 428, a glutamic acid at 97, anarginine at 97, a glutamic acid at 319, an arginine at 319, a glutamicacid at 358, an arginine at 358, a glutamic acid at 443, or an arginineat 443. In one embodiment, the alpha-amylase variant comprises asubstitution of an N193 or a V416 or both, e.g., a substitution of N193For V416G, or both. The alpha-amylase variant features deletion of aminoacids 179 and 180 in other embodiments.

In one embodiment of the method, the parent AmyS-like alpha-amylase isSEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 11, 12, 15, or 16.

The phytase can be added before or after the alpha-amylase variant. Thestarch slurry may be preincubated after adding the phytase, and beforeadding the alpha-amylase variant. In one embodiment, the inclusion ofthe phytase results in an increase in thermostability of thealpha-amylase variant relative to a comparable method that does notinclude contacting the starch slurry with phytase. In one embodiment ofthe method, the phytase and the alpha-amylase variant are present in asingle blend before adding to the starch slurry. In another, the phytasehas the amino acid sequence of SEQ ID NO: 31.

In a further aspect, this relates to a method for liquefying starch in aslurry comprising a substrate which includes plant material such asgranular starch from either a dry or wet milling process, the methodcomprising a primary and/or secondary liquefaction step, the methodcomprising adding to the slurry in the primary and/or secondaryliquefaction step, in any order, a combination of at least one phyticacid hydrolyzing enzyme and at least one alpha-amylase simultaneously orseparately. The method can further comprise saccharifying the liquefiedstarch to obtain fermentable sugars; and recovering the fermentablesugars. In some embodiments, the method further comprises fermenting thefermentable sugars under suitable fermentation conditions to obtainend-products such as alcohol. In some embodiments, the enzymecomposition contains at least one alpha-amylase and a phytase. In someembodiments, the enzyme composition is in blended form. In a furtheraspect, this relates to a method for fermenting a starch substrate, themethod comprising adding in any order a combination of an alpha-amylaseand a phytase in a single or split dose. In another aspect, the treatedstarch substrate is fermented to ethanol.

In various embodiments of the method, the reference alpha-amylase is SEQID NO: 1 or 2, and the alpha-amylase variant is a S242A, S242D, S242E,S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242T alpha-amylasevariant.

In another aspect is a method of obtaining a fermentable substrate bycontacting a slurry of milled grain containing granular starch, with aphytic acid-hydrolyzing enzyme at a temperature of about 0 to about 30°C. less than the starch gelatinization temperature, contacting theslurry with an alpha-amylase, raising the temperature above thegelatinization temperature for the granular starch to allowgelatinization of the starch, hydrolyzing the gelatinized starch bycontacting the gelatinized starch with the alpha-amylase for a timesufficient to hydrolyze the starch, and obtaining a fermentablesubstrate. The phytic acid hydrolyzing enzyme can be a bacterial orfungal phytase. The fungal phytase can be an Aspergillus phytase or aButtiauxella phytase. In some embodiments, the bacterial phytase is fromEscherichia coli.

Provided, then, are methods of producing a fermentable substrate from astarch-containing slurry comprising milled grain, the method comprisesthe steps of

-   -   a) contacting the starch-containing slurry with at least one        phytase and at least one alpha-amylase in an amount sufficient        to produce a fermentable substrate from the starch;

wherein the contact with the phytase and the alpha-amylase is initiatedat or about the same time, or separately in any order, and wherein thealpha-amylase is a variant amylase comprising an amino acid sequence atleast 95% identical to that of a parent AmyS-like alpha-amylase, andhaving a substitution at an amino acid position corresponding toposition 242 of a reference alpha-amylase, said variant havingdetectable alpha-amylase activity; and

b) incubating the starch slurry under conditions permissive of activityof the phytase and the alpha-amylase for a time that allows productionof the fermentable substrate; wherein when the contact with the phytaseis initiated before the amylase, the slurry is incubated at atemperature that is 0-30° C. less than the gelatinization temperatureprior to contacting the slurry with the amylase, after which thetemperature is raised above the gelatinization for a time effective tohydrolyze the starch.

In one embodiment, the reference alpha-amylase is SEQ ID NO: 1 or 2, andthe parent AmyS-like alpha-amylase comprises any of SEQ ID NOs: 1, 2, 6,7, 8, 9, 10, 11, 12, 15, or 16. The alpha-amylase variant is preferablya S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q,or S242T alpha-amylase variant.

In one embodiment, the method comprises use of at least one additionalenzyme which is a phytase, cellulase, protease, aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, chitinase, cutinase,cyclodextrin glucanotransferase, deoxyribonuclease, esterase,α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase,β-glucosidase, haloperoxidase, invertase, isomerase, laccase, lipase,mannosidase, oxidase, pectinase, peptidoglutaminase, peroxidase,polyphenoloxidase, nuclease, ribonuclease, transglutaminase, xylanase,pullulanase, isoamylase, carrageenase, or a combination of two or moreof the foregoing.

The method is preferably part of a process for starch degradation,liquefaction, fermentation, alcohol production, sweetener production,production of a fermentable substrate, cleaning, washing, stain removal,or baking process, or the like.

In another aspect, the disclosure relates to a process for producing afermentable sugar comprising a) mixing milled starch-containing materialwith water and thin stillage, wherein the thin stillage is in the rangeof 10 to 70% v/v and obtaining a slurry comprising starch and having adry solids (ds) content of 20 to 50% w/w, b) treating the slurry with aphytase prior to or simultaneously with liquefying the starch, c)liquefying the starch, d) adding an alpha-amylase to the starch eitherduring step b) and/or simultaneously with the liquefying step and e)saccharifying the liquefied starch to obtain fermentable sugars, whereinthe pH is not adjusted during any of the steps a), b), c), d) or e). Insome embodiments, the fermentable sugar is recovered and purified orisomerized. In other embodiments, the phytase is added prior to theliquefaction step. In further embodiments, the alpha-amylase is addedwith the phytase. In yet further embodiments, a second alpha-amylasedose is added during the liquefaction step.

Also provided herein are methods of method of treating astarch-containing material or a starch with an amylase. The methodscomprise the step of contacting the starch-containing material or thestarch with a composition comprising at least one alpha-amylase variantdisclosed herein under conditions sufficient to allow detectableactivity of the alpha-amylase; wherein the variant amylase any variantdisclosed herein; and wherein the starch is at least partially degradedby the variant amylase. In preferred embodiments, the alpha-amylasevariant is a S242A, S242D, S242E, S242F, S242G, S242H, S242L, S242M,S242N, S242Q, or S242T variant alpha-amylase. The methods areconveniently used as part of a process for starch degradation,liquefaction, fermentation, alcohol production, sweetener production,production of a fermentable substrate, cleaning, washing, stain removal,or baking process, or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows alignment of amino acid sequences among several candidateparent alpha-amylases (AmyS-like amylases) for use herein. Positionscorresponding to any amino acid position (e.g., 1 through 520) of theamylase from Geobacillus stearothermophilus (SEQ ID NO: 1) can bereadily determined. SEQ ID NO: 1, alpha-amylase from G.stearothermophilus “BSG”; SEQ ID NO: 2, truncated amylase from G.stearothermophilus (AmyS, SPEZYME XTRA); SEQ ID NO: 3, G.stearothermophilus (S242A variant amylase); SEQ ID NO: 4, G.stearothermophilus (S242Q variant amylase); SEQ ID NO: 5, G.stearothermophilus (S242E variant amylase); SEQ ID NO: 6, Yamane 707amylase; SEQ ID NO: 7, mature LAT amylase; SEQ ID NO: 8, Bacilluslicheniformis wild-type amylase [TERMAMYL (NOVOZYMES)=SEQ ID NO: 8 in WO02/10355A2]; SEQ ID NO: 9, B. amyloliquefaciens amylase, BAN; SEQ ID NO:10, STAINZYME=AA560 which is SEQ ID NO: 2 in WO 0060060 or SEQ ID NO: 24in U.S. Pat. No. 6,528,298; SEQ ID NO: 11, B. halmapalus amylase(NATALASE); SEQ ID NO: 12, KSM-1378 (KAO CORP., SEQ ID NO: 3 inEP1199356); SEQ ID NO: 13, Bacillus spp. KSM-K38 (KAO CORP., SEQ ID NO:4 in U.S. Pat. No. 6,403,355 B1); SEQ ID NO: 14, Bacillus spp. KSM-K36(KAO CORP., SEQ ID NO: 2 in U.S. Pat. No. 6,403,355 B1); SEQ ID NO: 15,LIQUOZYME SC (NOVOZYMES); SEQ ID NO: 16, Consensus Parent Alpha-AmylaseSequence #1;

FIG. 2 shows the pHPLT-AmyS plasmid.

FIG. 3 shows percent residual activity of S242 variants after heatstress at 95° C. for 30 minutes. A positive control, G.stearothermophilus with A179-180 with the C-terminus truncated by 29amino acids (i.e., SEQ ID NO: 2) is also shown. Lines indicate 2× and 3×above the standard deviation of the percent residual activity of thewild-type enzyme. S242A and S242Q clearly show higher residualactivities than the wild-type.

FIG. 4: Panels A, B, C, D, E, F, G, H, and I show pair-wise alignmentsand consensus sequences for several sequences from FIG. 1, and feature,respectively, Consensus Sequences 2, 3, 4, 5, 6, 7, 8, 9, and 10, or SEQID NOs: 22, 23, 24, 25, 26, 27, 28, 29, and 30, respectively.

FIG. 5 shows the thermal melting curves and the melting points for thewild-type and amylase variants without added calcium.

FIG. 6 shows the thermal melting curves and the melting points in thepresence of 2 mM added calcium for both the wild-type and the amylasevariants.

FIG. 7 shows the activity profile at 4, 10, and 20 minutes for SpezymeXtra and two variants, relative to Liquozyme SC.

FIG. 8 shows the activity profile of four variants relative to the S242Qvariant for three time points.

FIG. 9 shows the viscosity reduction of corn flour due to the action ofthe alpha-amylases Liquozyme SC, Spezyme Ethyl or Spezyme Xtra at a 30μg dose.

FIG. 10 shows the viscosity reduction of corn flour due to the action ofthe alpha-amylases Liquozyme SC or Spezyme Xtra, or one of two variants(S242A and S242Q) at a 30 μg dose.

FIG. 11 shows the viscosity reduction of corn flour due to the action ofthe alpha-amylase Liquozyme SC or Spezyme Xtra, or one of two variants(S242A and S242Q) at a 20 μg dose.

FIG. 12 shows the DE progression of whole ground corn treated withLiquozyme SC, Spezyme Xtra, or one of two variants (S242A and S242Q)over time (0, 30, 60 and 90 minutes). Dosing of liquefaction enzymespre- and post-jet are indicated as “X+Y” where: X and Y represent thenumber of units of enzyme added before and after the jet, respectively.

FIG. 13 shows the viscosity post-jet of whole ground corn treated withLiquozyme SC, Spezyme Xtra, or one of two variants (S242A and S242Q)over time (0, 30, 60 and 90 minutes). X and Y are as in FIG. 12.

FIG. 14 shows the DE progression of whole ground corn treated withphytase and an amylase (Spezyme Xtra or S242Q variant) over time (0, 30,60 and 90 minutes). MAXALIQ is a phytase/amylase blend available fromGenencor, a Danisco Division. Phytase effect was observed during primaryliquefaction using 242Q AA in low pH (5.2) liquefaction process of wholeground corn, 32% ds. corn containing 30% thin stillage, no jet cooking.Reference is made to Example 8.

FIG. 15 shows the viscosity post-jet of whole ground corn treated withphytase and amylase (e.g., Spezyme Xtra or S242Q variant) over time (0,30, 60 and 90 minutes). Conditions were as in FIG. 14. Reference is madeto Example 8.

FIG. 16 shows the DE progression of whole ground corn treated with theS242Q variant and phytase. Conditions included: 32% whole ground corncontaining 30% thin stillage, pH 5.2. During 30 min preincubation, BP-17phytase was added at 0 (control), 1, 2, 4, 6, 9, and 12 FTU, primaryliquefaction followed. Enzymes used during primary liquefaction: 242Q 4AAU/gm ds corn, incubation time was 45 min at 70° C. Secondaryliquefaction was at 90° C. for 90 min. Reference is made to Example 9.

FIG. 17 shows the viscosity post-jet of whole ground corn treated withthe S242Q variant and phytase. Effects of phytate removal on theviscosity reduction of during batch liquefaction of whole ground corn at90° C., pH 5.2 were observed. Pre-incubation with phytase was as in FIG.16. Reference is made to Example 9.

FIG. 18 shows the effect of phytase treatment of whole ground corn onthe increase in the thermostability and low pH stability of the S242Qvariant. The effects of low pH on the liquefaction process of wholeground corn, 32% ds. corn containing 30% thin stillage were observed.Triangles, pH 5.2; squares, pH 4.8; diamonds, pH 4.5; and circles, pH4.2. Reference is made to Example 9.

FIG. 19 shows the effect of phytase addition during primary liquefactionof whole ground corn on the viscosity reduction after jet cooking.Conditions were as in FIG. 18. White, pH 5.2; black, pH 4.8; stippled,pH 4.5; and hashed, pH 4.2. Reference is made to Example 9.

FIG. 20 is a graph showing the effects of BP-17 concentration duringprimary liquefaction of whole ground corn, pH 5.2 by S242Q variant AA(4AAU/gds corn) on the rate of DE progression at 90° C. were observed.The rates of DE progression (squares) and the percent phytic acidreduction as IP6 (diamonds) were measured.

FIG. 21 is a graph-showing the effect of the S242Q alpha-amylase varianton DE progression under conventional processing conditions. Observingeffects of pH on S242Q performance in liquefaction with jet cooking (32%DS, 30% thin stillage, 10 min slurry at 85° C.+107° C. jet+3 minresidence time+90 min secondary batch liquefaction at 85° C.). Squares,pH 5.8; diamonds, pH 5.5. Reference is made to Example 8.

FIG. 22 is a graph depicting the performance of S242Q (filled circles)and its variants (open circles), as a function of charge, in the ricestarch microswatch assay under North American laundry conditions usingS242Q combinatorial charge library, rice starch microswatch cleaning inTide 2×, at 20° C. Reference is made to Example 16.

FIG. 23 is a graph depicting the performance of a truncated Bacillus sp.TS-23 amylase (closed circles) with the following mutations: Q98R,M201L, S243Q R309A, Q320R, Q359E, and K444E and its charge variants(open circles) (see co-pending U.S. Patent Application No.PCT/US2008/007103, filed 6 Jun. 2008) in the rice starch microswatchassay as a function of charge under Western European laundry conditionswith TS23t combinatorial charge library, rice starch microswatchcleaning in Persil at 40° C. Reference is made to Example 16.

FIG. 24 is a graph depicting the performance of S242Q (closed circles)and its variants (open circles) in the BODIPY-starch assay as a functionof charge. S242Q combinatorial charge library (CCL), specific activityon BODIPY-starch, standard assay conditions Reference is made to Example16.

FIG. 25 A is a graph depicting the relative BODIPY-starch hydrolysis asa function of relative shake tube expression (i.e., relativeBODIPY-starch hydrolysis vs. relative shake tube expression). FIG. 25Bis a graph depicting the relative microswatch-starch hydrolysis as afunction of relative shake tube expression (i.e., relativemicroswatch-starch hydrolysis vs. relative shake tube expression).Reference is made to Example 19.

FIG. 26A is a graph depicting the relative shake tube expression as afunction of charge. FIG. 26B is a graph depicting the relativeBODIPY-starch hydrolysis as a function of charge. Reference is made toExample 19.

FIG. 27A is a graph depicting the relative shake tube expression as afunction of charge. FIG. 27B is a graph depicting the relativemicroswatch cleaning activity as a function of charge. Reference is madeto Example 19.

DETAILED DISCLOSURE

1. Definitions & Abbreviations

In accordance with this disclosure, the following abbreviations anddefinitions apply. It should be noted that as used herein, the singularforms “a,” “an,” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “apolypeptide” includes a plurality of such polypeptides and reference to“the formulation” includes reference to one or more formulations andequivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. The following terms are provided below.

1.1. Abbreviations

The following abbreviations apply unless indicated otherwise:

AATCC American Association of Textile Chemists and Colorists; ADWautomatic dish washing; AE alcohol ethoxylate; AEO alcohol ethoxylate;AEOS alcohol ethoxysulfate; AES alcohol ethoxysulfate; AFAU acid fungalalpha-amylase units; AGU glucoamylase activity units; AOSα-olefinsulfonate; AS alcohol sulfate; BAA bacterial alpha-amylase; ° C.degrees Centigrade; CCL combinatorial charge library; cDNA complementaryDNA; CMC carboxymethylcellulose; dE total color difference, as definedby the CIE-LAB color space; dH₂O deionized water; dIH₂O deionized water,Milli-Q filtration; DE Dextrose Equivalent; DNA deoxyribonucleic acid;dNTP deoxyribonucleotide triphosphates; DO dissolved oxygen; DP3 degreeof polymerization with three subunits; DPn degree of polymerization withn subunits; DS (or ds) dry solids content; DSC differential scanningcalorimetry; DTMPA diethyltriaminepentaacetic acid; EC enzyme commissionfor enzyme classification; EDTA ethylenediaminetetraacetic acid; EDTMPAethylenediaminetetramethylene phosphonic acid; EO ethylene oxide; eqequivalents; ETOH ethanol; F&HC fabric and household care; FTU “fitase”units, phytate hydrolyzing unit; g (or gm) grams; GAU glucoamylase unit;gpg grains per gallon; g/l grams per liter; Genencor Danisco U.S. Inc,Genencor Division, Palo Alto, CA; H₂O water; HDG heavy duty granulardetergent; HDL heavy duty liquid detergent; HFCS high-fructose cornsyrup; HFSS high-fructose starch-based syrup; HPAEC-PAD high performanceanion exchange chromatography with pulsed amperometric detection; hr(s)hour/hours; IKA IKA Works Inc. 2635 North Chase Parkway SE, Wilmington,NC; IPTG isopropyl β-D-thiogalactoside; JPN Japan; kg kilograms; LALuria Agar; LAS linear alkylbenezenesulfonate; LB Luria Broth; LU LipaseUnits; M molar; MBD medium MOPS-based defined medium; MES2-(N-morpholino)ethanesulfonic acid; mg milligrams; min(s)minute/minutes; mL (or ml) milliliters; mm millimeters; mM millimolar;MOPS 3-(N-Morpholino)-propanesulfonic acid; MW molecular weight; NANorth America; Ncm Newton centimeter; NEO neomycin; ng nanogram; nmnanometer; NOBS nonanoyloxybenzenesulfonate; N Normal; NTAnitrilotriacetic acid; PAHBAH p-hydroxybenzoic acid hydrazide; PCRpolymerase chain reaction; PEG polyethyleneglycol; pI isoelectric point;ppm parts per million; PVA poly(vinyl alcohol); PVPpoly(vinylpyrrolidone); RAU Reference Amylase Units; RMS root meansquare; RNA ribonucleic acid; rpm revolutions per minute; SAPUspectrophotometric acid protease unit; SAS secondary alkane sulfonates;1X SSC 0.15M NaCl, 0.015M sodium citrate, pH 7.0; sec seconds; % SRIpercent stain removal index; SSF simultaneous saccharification andfermentation; TAED tetraacetylethylenediamine; T_(m) thermal midpointfor a DSC curve, or melting tem- perature of a protein; TNBStrinitrobenzenesulfonic acid; μg micrograms; μl, (μL) microliters; μNmmicroNewton meters; μm micrometer; μM micromolar; U units; V/V volume tovolume; WE Western Europe; wt % weight percent; w/v (or W/V)weight/volume; w/w (or W/w) weight/weight; wt wild-type.

1.2. Definitions

In some aspects, the present disclosure relies on routine techniques andmethods used in the field of genetic engineering and molecular biology.The following resources include descriptions of general methodologyuseful in accordance with what is disclosed herein: Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL (2^(nd) Ed., 1989); Kreigler,GENE TRANSFER AND EXPRESSION; A LABORATORY MANUAL (1990) and Ausubel etal., Eds. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1994).

These general references provide definitions and methods known to thosein the art. Unless defined otherwise herein, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which the disclosurepertains. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY, 2D ED., John Wiley and Sons, New York (1994) and Hale &Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY(1991) provide one of skill with general dictionaries of many of theterms used in this disclosure.

“Isolated” means that the isolated substance, e.g. a compound or asequence, is modified by the hand of man relative to that compound orsequence as found in nature. For example, an isolated sequence is atleast partially free, or even substantially free, from at least oneother component with which the sequence is naturally associated as foundin nature.

“Purified” when used to describe a material or substance means that thematerial or substance is in a relatively pure state, e.g., at leastabout 90% pure, at least about 95% pure, at least about 98% pure, or atleast about 99% pure.

As used herein, “starch” refers to any carbohydrate compositioncomprising complex polysaccharides, comprising amylose and/oramylopectin with the formula (C₆H₁₀O₅), wherein “X” can be any number.Preferably, starch refers to any such carbohydrate that is naturallypresent in plants, including but not limited to grains, grasses, tubers,and roots, and more specifically from wheat, barley, corn, rye, rice,sorghum, cassaya, millet, potato, sweet potato, and tapioca. Starch canalso refer to synthetic starches or modified starches, such aschemically-modified starch for use as a detectable substrate for enzymeassays, or starches chemically- or enzymatically-modified to improve oneor more properties for use.

As used herein, “phytic acid” (or inositol hexakisphosphate (IP6)), isthe principle storage form of phosphorus in many plant tissues, such asbran, seeds, and the like. Phytic acid is also referred to as “phytate”herein, especially when in salt form. Various other inositol phosphatessuch as inositol penta- (IP5), tetra- (IP4), and triphosphate (IP3) arealso referred to herein as phytates. Phytates are generally indigestibleby man and most monogastric animals.

Enzymes that degrade phytates are referred to herein as “phytases” or“fytases” are generally myo-inositol-hexaphosphate phosphohydrolases.Phytase activity is defined as fytase units (FTU or U), where one FTU isdefined as the quantity of enzyme that liberates 1 micromol ofinorganic-P per minute from 0.0015 mol/l sodium phytate at pH 5.5, and37° C. This definition provides a useful measure of quantity of phytaseactivity and represents a simple bench mark measurement.Phytate-degrading enzymes of yeasts (e.g., Schwanniomyces occidentalis,Pichia anomala, Arxula adeninivorans), gram-negative bacteria (e.g.,Escherichia coli, Pseudomonas spp., Klebsiella spp.), and gram-positive(e.g., Bacillus spp.) have been identified and characterized. Phytasesfrom many plants, and from filamentous fungi such as Penicillium spp.,Aspergillus spp., Trichoderma spp. Mucor piriformis, and Cladosporiumspp., are also known. 3-phytases (EC 3.1.3.8) and 6-phytases (EC3.1.3.26), depending on the site of initiation of hydrolysis, have beencharacterized. Also, phytase have been characterized, based on their pH“optima,” as either acid (pH optima around 5) or alkaline (pH optimaaround 9). A variety of commercial phytases are available, includingROVABIO (Genencor International).

“Amylase” refers to an enzyme that is capable of catalyzing the cleavageof a starch substrate, leading to a degradation or partial degradationof the starch. Amylases are generally hydrolases that cleave glycosidiclinkages in starch. As used herein amylase includes any glucoamylase,alpha-amylase, β-amylase, for example, the wild-type alpha-amylases ofBacillus spp., especially B. licheniformis. Generally, alpha-amylases(EC 3.2.1.1; α-D-(1→4)-glucan glucanohydrolase) are endo-acting enzymesdefined as cleaving α-D-(1→4) β-glycosidic linkages within the starchmolecule in a random fashion. In contrast, the exo-acting amylolyticenzymes, such as β-amylases (EC 3.2.1.2; α-D-(1→4)-glucanmaltohydrolase) and some product-specific amylases like maltogenicalpha-amylase (EC 3.2.1.133) cleave the substrate starch molecule fromthe non-reducing end. β-Amylases, α-glucosidases (EC 3.2.1.20;α-D-glucoside glucohydrolase), glucoamylase (EC 3.2.1.3;α-D-(1→4)-glucan glucohydrolase), and product-specific amylases canproduce malto-oligosaccharides of specific length from starch. Wild-typealpha-amylase from Bacillus stearothermophilus or “AmyS” amylase issometimes referred to herein as XTRA or SPEZYME XTRA, which arecommercial AmyS products from Genencor International.

As used herein, “AmyS-like alpha-amylases” are useful as parent amylasesherein. AmyS-like alpha-amylases constitute a class of alpha-amylasesherein, based on the substantial homology found between them. “AmyS-likealpha-amylase” is intended to indicate the class of alpha-amylases, inparticular Bacillus alpha-amylases, especially Geobacillusstearothermophilus alpha-amylases, which, at the amino acid level,exhibit a substantial identity to the alpha-amylase having the aminoacid sequence shown in SEQ ID NO: 2, herein. Spezyme Xtra iscommercially available from Danisco US Inc, Genencor Division.Geobacillus stearothermophilus has been referred to as Bacillusstearothermophilus in the literature and the two may be usedinterchangeably herein. All the alpha-amylases having the amino acidsequences provided herein as SEQ ID NOS: 1, 6, 7, 8, 9, 10, 11, 12, 15and 16, respectively, are considered to be AmyS-like alpha-amylases andthus are suitable as parent alpha-amylases. AmyS-like alpha-amylasesalso include alpha-amylases i) having amino acid sequences with at least60% homology (identity), such as at least 70%, at least 75%, or at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity, with at least one of theamino acid sequences shown in SEQ ID NOS: 1, 6, 7, 8, 9, 10, 11, 12, 15and 16, and/or ii) that are encoded by a DNA sequence that hybridizeswith a DNA sequence encoding any of the above-specified alpha-amylases,or those apparent from SEQ ID NOS: 9 (BAN), 5 (BSG), 3 (SP722), 1(SP690), 7 (LAT), 11 (AA560) of WO 06/002643 or of the presentspecification, which encode any of the amino acid sequences shown in SEQID NOS: 1, 6, 7, 8, 9, 10, 11, 12, 15 and 16 herein, respectively. Stillfurther homologous alpha-amylases useful as AmyS-like alpha-amylases andthus, as parent enzymes for producing variants herein, include thealpha-amylase produced by the B. licheniformis strain described in EP0252666. (ATCC 27811) and the alpha-amylases identified in WO 91/00353and WO 94/18314. Commercial AmyS-like alpha-amylases are comprised inthe products sold under the following trade names: Spezyme® AA andULTRAPHLOW (available from Danisco US Inc, Genencor Division), andKeistase™ (available from Daiwa) and LIQUEZYME SC (available fromNovozymes, Denmark). Section 1.5 herein below provides furtherinformation regarding AmyS-like alpha-amylases. Table A therein providesa list of several useful AmyS-like alpha-amylases, as well as aconvenient method of comparing amino acid sequence identities therebetween. The skilled artisan will appreciate the similar tables can beconstructed for other alpha-amylases to determine their suitability foruse herein as apparent enzyme.

As used herein, “spectrophotometric acid protease unit” (“SAPU”) is aunit of protease enzyme activity, wherein in 1 SAPU is the amount ofprotease enzyme activity that liberates one micromole of tyrosine perminute from a casein substrate under conditions of the assay.

“Glucoamylase unit” (“GAU”), is a measure of amylolytic activity definedas the amount of enzyme activity that will produce 1 g of reducingsugar, calculated as glucose, per hour from a soluble starch substrateat pH 4.2 and 60° C.).

As used herein, the term “variant” may be used interchangeably with theterm “mutant.” “Variants” can refer to either polypeptides or nucleicacids. Variants include one or more sequence “modifications,” which asused herein include substitutions, insertions, deletions, truncations,transversions, and/or inversions, at one or more locations relative to areference sequence. Each modifications can include changes that resultin a change of one or more amino acid residues or nucleotides in asequence, relative to the reference sequence. Variant nucleic acidsinclude sequences that are complementary to sequences that are capableof hybridizing to the nucleotide sequences presented herein. Forexample, a variant nucleic acid sequence herein can be at leastpartially complementary to a sequence capable of hybridizing understringent conditions (e.g., 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl, 0.015M sodium citrate, pH 7.0}) to a nucleotide sequences presented herein.More preferably, the term variant encompasses sequences that arecomplementary to sequences that are capable of hybridizing under highlystringent conditions (e.g., 65° C. and 0.1×SSC) to the nucleotidesequences presented herein.

“Thermostable” when used to describe an enzyme means the enzyme is morethermostable than a reference enzyme. In the present application, analpha-amylase variant is more thermostable than a wild-type B.licheniformis alpha-amylase if the variant has a relatively higherenzymatic activity after a specific interval of time under the sameexperimental conditions, e.g., the same temperature, substrateconcentration, etc. Alternatively, a more thermostable enzyme has ahigher heat capacity determined by differential scanning calorimetry,compared to a reference enzyme.

“Melting temperature” (T_(m)) of a polypeptide is a temperature at whichthe conformation of the polypeptide undergoes a measurabletemperature-dependent change. Protein conformation and T_(m) can beanalyzed, for example, by circular dichroism, one of the most generaland basic tools to study protein folding. Circular dichroismspectroscopy measures the absorption of circularly polarized light. Inproteins, structures such as alpha helices and beta sheets are generallychiral, and thus absorb circularly polarized light. The light absorptionprovides a measure of the degree of foldedness of the protein. Changesin this absorption as a function of temperature or concentration of asequence, or that the cell is derived from a cell so modified oraltered. Thus, for example, recombinant cells may express genes that arenot found within the native (non-recombinant) form of the cell or mayexpress native genes that are otherwise differently expressed (e.g.under-expressed, or over-expressed), abnormally expressed, or notexpressed at all.

As used herein, “nucleotide sequence” or “nucleic acid sequence” refersto any sequence of two or more nucleotides, ribonucleotides, or thelike, or derivatives thereof. Nucleotide sequences includeoligonucleotide and polynucleotide sequences, as well as variants,homologues, fragments and derivatives thereof. A nucleotide sequence maybe single-, double-, or multi-stranded. The nucleotide sequence may befrom any source or origin, e.g., genomic, synthetic, or recombinant, andincludes genomic DNA, cDNA, synthetic DNA, and RNA, and the like as wellas hybrids thereof. Nucleotide sequences may comprise one or more codonsand may encode one or more polypeptides. Nucleotide sequences maypreferentially assume one or more energetically preferredthree-dimensional structures.

A “vector” refers to a nucleotide sequence frequently useful forexperimental use in vitro, or for introduction of nucleic acids into oneor more cell types. Vectors include cloning vectors, in vivo or in vitroexpression vectors, shuttle vectors, plasmids, phagemids, cosmids, phageparticles, cassettes and the like.

An “expression vector” as used herein means a DNA construct comprising aDNA sequence which is operably-linked to a suitable control sequencecapable of effecting expression of the DNA in a suitable host. Suchcontrol sequences may include a promoter to effect transcription, anoptional operator sequence to control transcription, a sequence encodingsuitable ribosome binding sites on the mRNA, enhancers and sequenceswhich control termination of transcription and translation.

A polynucleotide or a polypeptide having a certain percent (e.g., atleast about 80%, 85%, 90%, 95%, or 99%) of sequence identity withanother sequence means that, when aligned, that percentage of bases oramino acid residues are the same in comparing the two sequences. Thisalignment and the percent homology or identity can be determined usingany suitable software program known in the art, for example thosedescribed in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel etal. (eds) 1987, Supplement 30, section 7.7.18). Such programs mayinclude the GCG Pileup program, FASTA (Pearson et al. (1988) Proc. Natl,Acad. Sci. USA 85: 2444-2448), and BLAST (BLAST Manual, Altschul et al.,Natl Cent. Biotechnol. Inf., Natl Lib. Med. (NCIB NLM NIH), Bethesda,Md., and Altschul et al., (1997) NAR 25:3389-3402). Another alignmentprogram is ALIGN Plus (Scientific and Educational Software, PA), usingdefault parameters. Another sequence software program that finds use isthe TFASTA Data Searching Program available in the Sequence SoftwarePackage Version 6.0 (Genetics Computer Group, University of Wisconsin,Madison, Wis.).

One skilled in the art will recognize that sequences encompassed by thedisclosure are also defined by the ability to hybridize under stringenthybridization conditions with the exemplified amyS sequence (e.g., SEQID NO:5 of WO 06/002643). A nucleic acid is hybridizable to anothernucleic acid sequence when a single stranded form of the nucleic acidcan anneal to the other nucleic acid under appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known in the art (see, e.g., Sambrook (1989) supra,particularly chapters 9 and 11). In some embodiments, stringentconditions correspond to a T_(m) of 65° C. and 0.1×SSC, 0.1% SDS.

A “gene” refers to a DNA segment that is involved in producing apolypeptide and includes regions preceding and following the codingregions as well as intervening sequences (introns) between individualcoding segments (exons).

“Heterologous” with reference to a polynucleotide or protein refers to apolynucleotide or protein that does not naturally occur in a host cell.In some embodiments, the protein is a commercially important industrialprotein. It is intended that the term encompass proteins that areencoded by naturally occurring genes, mutated genes and nucleic acidsencoding heterolorougs proteins such as fusion proteins, and/orsynthetic genes.

“Endogenous” with reference to a polynucleotide or protein refers to apolynucleotide or protein that occurs naturally in the host cell.

As used herein, “transformed”, “stably transformed”, and “transgenic”used in reference to a cell means the cell comprises at least onenon-native (e.g., heterologous) nucleic acid sequence. Astably-transformed cell comprises at least one such nucleic acidsequence integrated into its genome, or in an episomal plasmid that ismaintained through multiple generations.

As used herein, “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

A “signal sequence” means a sequence of amino acids covalently-bound tothe N-terminal portion of a protein, which facilitates the transport ofthe protein, e.g., secretion of the mature form of the protein outsidethe cell. The definition of a signal sequence is functional. The matureform of the extracellular protein lacks the signal sequence which iscleaved off, e.g., during the secretion process.

As used herein, the term “derived” encompasses the terms “originatedfrom”, “obtained from” or “obtainable from”, and “isolated from”.

The terms “protein” and “polypeptide” are used interchangeably herein.The conventional one-letter or three-letter code for amino acid residuesis used herein.

A “promoter” is a regulatory sequence that is involved in binding RNApolymerase to initiate transcription of a gene. The promoter may be aninducible promoter or a constitutive promoter. For example, cbh1 fromTrichoderma reesei, an inducible promoter, can be used herein.

“Operably-linked” refers to juxtaposition wherein elements are in anarrangement allowing them to be functionally related, even where not inclose physical proximity. For example, a promoter is operably-linked toa coding sequence if it is capable of controlling the coding sequenceand does control the transcription of the sequence under conditionspermissive thereof, or conducive thereto.

“Selective marker” refers to a gene capable of expression in a host, andwhich allows selecting those hosts expressing the marker gene. Examplesof selectable markers include but are not limited to gene that providealtered resistance to an antimicrobial agent (e.g., hygromycin,bleomycin, or chloramphenicol) and/or genes that confer metabolicselectivity, for example, a nutritional advantage on the host cell, suchas growth on a particular substrate as a sole source of carbohydrate.

“Introduced” in the context of inserting a nucleic acid sequence into acell, means “transfection”, or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid sequence intoa eukaryotic or prokaryotic cell wherein the nucleic acid sequence maybe incorporated into the genome of the cell (e.g., chromosome, plasmid,plastid, or mitochondrial DNA), converted into an autonomous replicon,or transiently expressed (e.g., transfected mRNA).

“Host,” “host strain,” or “host cell” means a suitable cell in which toplace an expression vector or DNA construct comprising a polynucleotide,e.g., encoding a variant alpha-amylase. Host strains are preferablybacterial cells. In a preferred embodiment, “host cell” means cellsand/or protoplasts created from the cells of a microbial strain, e.g., aBacillus spp.

The term “culturing” refers to growing a population of microbial cellsunder suitable conditions in a medium capable of supporting such growth.In one embodiment, culturing refers to fermentative bioconversion of astarch substrate containing granular starch to an end-product (typicallyin a vessel or reactor).

“Fermentation” is the breakdown of organic substances by microorganismsto produce simpler organic compounds. While fermentation generallyoccurs under predominantly anaerobic conditions, it is not intended thatthe term be limited to strict anaerobic conditions, as fermentation alsooccurs in the presence of oxygen.

The term “enzymatic conversion” in general refers to the modification ofa substrate by enzyme action. The term as used herein also refers to themodification of a starch substrate by the action of an enzyme.

As used herein the term “saccharification” refers to enzymaticconversion of starch to glucose.

The term “gelatinization” means at least partial solubilization of astarch granule or molecule, e.g., by cooking to form a viscoussuspension.

The term “liquefaction” generally refers to a stage during starchconversion in which starch is at least partially hydrolyzed to give alower molecular weight product, e.g., soluble dextrins.

The term “degree of polymerization (DP)” refers to the number (n) ofanhydroglucopyranose units in a given saccharide. Examples of DP1 aremonosaccharides, such as glucose and fructose. Examples of DP2 aredisaccharides, such as maltose and sucrose. A DP>3 denotes polymers witha degree of polymerization of greater than 3. The skilled artisan willunderstand that compounds with greater DE are more polymeric.

“End-product” or “desired end-product” refer to any intended product ofan enzymatic reaction, e.g. a starch-derived molecule that isenzymatically converted from the starch substrate.

As used herein “dry solids content (ds)” refers to the total solids of aslurry in % on a dry weight basis. The term “slurry” refers to anaqueous mixture containing insoluble solids.

The term “residual starch” refers to any remaining starch (soluble orinsoluble) left in a composition after fermentation of astarch-containing substrate.

“A recycling step” refers to the recycling of mash components, which mayinclude residual starch, enzymes and/or microorganisms to fermentsubstrates comprising starch.

The term “mash” refers to a mixture of a fermentable carbon source(carbohydrate) in water used to produce a fermented product, such as analcohol. In some embodiments, the term “beer” and “mash” are usedinterchangeability.

“Stillage” means a mixture of non-fermented solids and water, such asthe residue after removal of alcohol from a fermented mash.

The terms “distillers dried grain (DDG)” and “distillers dried grainwith solubles (DDGS)” refer to a useful by-product of grainfermentation.

As used herein “ethanologenic microorganism” refers to a microorganismwith the ability to convert a carbohydrate (e.g., mono-, di-, oligo-, orpolysaccharides) to ethanol. The ethanologenic microorganisms areethanologenic by virtue of their ability to express one or more enzymesthat individually or collectively convert the carbohydrate to ethanol.

As used herein, “ethanol producer” or ethanol producing microorganism”refers to any organism or cell that is capable of producing ethanol froma hexose or pentose. Generally, ethanol-producing cells contain analcohol dehydrogenase and a pyruvate decarboxylase. Examples of ethanolproducing microorganisms include fungal microorganisms such as yeast.

As used herein, “specific activity” means an enzyme unit defined as thenumber of moles of substrate converted to product by an enzymepreparation per unit time under specific conditions. Specific activityis expressed as units (U)/unit weight of protein, generally, U/mgprotein.

“Yield” refers to the amount of end-product or desired end-productsproduced using the methods of the present disclosure. In someembodiments, the yield is greater than that produced using methods knownin the art. In some embodiments, the term refers to the volume of theend product and in other embodiment the term refers to the concentrationof the end product.

As used herein, “biologically-active” refers to a compound or sequencethat has a measurable effect on a biological system, e.g., a cell, anorgan, or an organism.

“ATCC” refers to American Type Culture Collection located at Manassas,Va. 20108 (ATCC).

“NRRL” refers to the Agricultural Research Service Culture Collection,National Center for Agricultural Utilization Research (and previouslyknown as USDA Northern Regional Research Laboratory), Peoria, Ill.

As used herein, “food” means any ingredient, component or compositionthat provides a nutritive value for an animal, including a human.

As used herein, by convention, when describing proteins and genes thatencode them, the term for the gene is generally italicized, (e.g., thegene that encodes amyL (B. licheniformis AA) may be denoted as amyL).The term for the protein is generally not italicized and the firstletter is generally capitalized, (e.g., the protein encoded by the amyLgene may be denoted as AmyL or amyL). Unless otherwise indicated,nucleic acid sequences are presented left to right in 5′ to 3′orientation, and amino acid sequences are written left to right in aminoto carboxy orientation, respectively.

As used herein the term “comprising” and its cognates are used in theirinclusive sense; that is, equivalent to the term “including” and itscorresponding cognates. Numeric ranges are inclusive of the numbersdefining the range.

The headings provided herein are not limitations of the various aspectsor embodiments of what is disclosed.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of that which isdisclosed, certain presently preferred methods and materials aredescribed with no intention to limit the practitioner to any particularmethods, protocols, and reagents described, as these may be varied. Allpatents and publications, including all sequences disclosed within suchpatents and publications, referred to herein are expressly incorporatedby reference.

2. Nomenclature

In the present description and claims, the conventional one-letter andthree-letter codes for amino acid residues are used. For ease ofreference, alpha-amylase variants are generally described by use of thefollowing nomenclature:

-   -   Original amino acid(s): position(s): substituted amino acid(s)

According to this nomenclature, for instance the substitution of serineby an alanine in position 242 is shown as:

-   -   Ser242Ala or S242A        a deletion of alanine in position 30 is shown as:    -   Ala30* or A30* or ΔA30        and insertion of an additional amino acid residue, such as        lysine, is shown as:    -   Ala30AlaLys or A3 OAK

A deletion of a consecutive stretch of amino acid residues, such asamino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).

Where a specific alpha-amylase contains a “deletion” in comparison withother alpha-amylases and an insertion is made in such a position this isindicated as:

-   -   *36Asp or *36D        for insertion of an aspartic acid in position 36.

Multiple mutations are separated by plus signs, i.e.:

-   -   Ala30Asp+Glu34Ser or A30N+E34S        representing mutations in positions 30 and 34 substituting        alanine and glutamic acid for asparagine and serine,        respectively.

When one or more alternative amino acid residues may be inserted in agiven position it is indicated as:

-   -   A30N,E or alternatively, A30N or A30E

Furthermore, when a position suitable for modification is identifiedherein without any specific modification being suggested, it is to beunderstood that any amino acid residue may be substituted for the aminoacid residue present in the position. Thus, for instance, when amodification of an alanine in position 30 is mentioned, but notspecified, it is to be understood that the alanine may be deleted orsubstituted for any other amino acid, i.e., any one of:

R, N, D, A, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

Further, “A30X” means any one of the following substitutions: A30R,A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K, A30M, A30F,A30P, A30S, A30T, A30W, A30Y, or A30V; or in short:

A30R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

If the parent enzyme—used for the numbering—already has the amino acidresidue in question suggested for substitution in that position thefollowing nomenclature is used:

“X30N” or “X30N,V” in the case where, for instance, one or N or V ispresent in the wild-type. This indicates that other corresponding parentenzymes are substituted to an “Asn” or “Val” in position 30.

3. Characteristics of Amino Acid Residues

Charged Amino Acids:

Asp, Glu, Arg, Lys, His

Negatively Charged Amino Acids (with the Most Negative Residue First):

Asp, Glu

Positively Charged Amino Acids (with the Most Positive Residue First):

Arg, Lys, His

Neutral Amino Acids:

Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Met, Cys, Asn, Gln, Ser, Thr,Pro

Hydrophobic Amino Acid Residues (with the Most Hydrophobic ResidueListed Last):

Gly, Ala, Val, Pro, Met, Leu, Ile, Tyr, Phe, Trp,

Hydrophilic Amino Acids (with the Most Hydrophilic Residue Listed Last):

Thr, Ser, Cys, Gln, Asn

4. Alpha-Amylases and AmyS-Like Amylases

4.1 Amino Acid Identities of Various Alpha-Amylase

A number of alpha-amylases produced by Bacillus spp. are highlyhomologous (identical) on the amino acid level and may be useful asparent enzymes herein. The percent identity (based on amino acidsequence) of a number of known Bacillus alpha-amylases, relative to eachother can be found in the below Table A:

TABLE A Amino acid sequence identity of several known Bacillusalpha-amylases 707 AP1378 BAN BSG SP690 SP722 AA560 LAT 707 100.0 86.466.9 66.5 87.6 86.2 95.5 68.1 AP1378 86.4 100.0 67.1 68.1 95.1 86.6 86.069.4 BAN 66.9 67.1 100.0 65.6 67.1 68.8 66.9 80.7 BSG 66.5 68.1 65.6100.0 67.9 67.1 66.3 65.4 SP690 87.6 95.1 67.1 67.9 100.0 87.2 87.0 69.2SP722 86.2 86.6 68.8 67.1 87.2 100.0 86.8 70.8 AA560 95.5 86.0 66.9 66.387.0 86.8 100.0 68.3 LAT 68.1 69.4 80.7 65.4 69.2 70.8 68.3 100.0

The skilled artisan will appreciate that percent identities can bedetermined from the literature, or by any means disclosed herein orknown in the art. For instance, the B. licheniformis alpha-amylase (LAT)(SEQ ID NO: 7) has been found to be about 81% homologous with the B.amyloliquefaciens alpha-amylase (SEQ ID NO: 9) and about 65% homologouswith the G. stearothermophilus alpha-amylase (BSG) (SEQ ID NO: 1).Further homologous alpha-amylases include SP690 and SP722 disclosed inWO 95/26397, as well as the #707 alpha-amylase derived from Bacillusspp. (SEQ ID NO: 6), described by Tsukamoto et al., Biochemical andBiophysical Research Communications, 151 (1988), pp. 25-31. The KSMAP1378 alpha-amylase is disclosed in WO 97/00324 (from KAO Corporation).

4.2 Parent Alpha-Amylases

AmyS-like alpha-amylases, as defined above, may be used as a parentalpha-amylase. In a preferred embodiment, the parent alpha-amylase isderived from G. stearothermophilus, e.g., one of those referred toabove, such as the G. stearothermophilus alpha-amylase having the aminoacid sequence shown in SEQ ID NO: 1 or 2.

4.3 Parent Hybrid Alpha-Amylases

The parent alpha-amylase (i.e., backbone alpha-amylase) may also be ahybrid alpha-amylase, i.e., an alpha-amylase that comprises acombination of partial amino acid sequences derived from at least twoalpha-amylases.

The parent hybrid alpha-amylase may be one, which on the basis of aminoacid homology (identity) and/or DNA hybridization (as defined above) canbe determined to belong to the AmyS-like alpha-amylase family describedabove. In such a case, the hybrid alpha-amylase is typically composed ofat least one part of a AmyS-like alpha-amylase and part(s) of one ormore other alpha-amylases selected from AmyS-like alpha-amylases ornon-AmyS-like alpha-amylases of microbial (bacterial or fungal) and/ormammalian origin.

Thus, the parent hybrid alpha-amylase may comprise a combination ofpartial amino acid sequences deriving from at least two AmyS-likealpha-amylases, or from at least one AmyS-like and at least onenon-AmyS-like bacterial alpha-amylase, or from at least one AmyS-likeand at least one fungal alpha-amylase. The AmyS-like alpha-amylase fromwhich a partial amino acid sequence derives, may be any of the specificAmyS-like alpha-amylase referred to herein.

For instance, the parent alpha-amylase may comprise a C-terminal part ofan alpha-amylase derived from a strain of B. licheniformis, and aN-terminal part of an alpha-amylase derived from a strain of G.stearothermophilus or from a strain of G. stearothermophilus (BSG).

5. Homology (Identity)

Homology may be determined as the degree of identity between twosequences indicating a relationship there between, e.g. a derivation ofthe first sequence from the second or vice versa. The homology may bedetermined by visual inspection or manual calculations, but moreconveniently by means of computer programs known in the art, such asGAP, a program provided in the GCG program package (described above).Thus, Gap GCG v8 may be used, for example with the default scoringmatrix for identity and the following default parameters: GAP creationpenalty of 5.0 and GAP extension penalty of 0.3, respectively fornucleic acidic sequence comparison, and GAP creation penalty of 3.0 andGAP extension penalty of 0.1, respectively, for protein sequencecomparison. GAP uses the method of Needleman and Wunsch, (1970), J. Mol.Biol. 48: 443-453, to make alignments and to calculate the identity.

A structural alignment between Spezyme Xtra (SEQ ID NO: 2) and, e.g.,another alpha-amylase may be used to identify equivalent/correspondingpositions in other AmyS-like alpha-amylases. One method of obtainingsaid structural alignment is to use the Pile Up program from the GCGpackage using default values of gap penalties, i.e., a gap creationpenalty of 3.0 and gap extension penalty of 0.1. Other structuralalignment methods include the hydrophobic cluster analysis (Gaboriaud etal., FEBS Lett. 224: 149-155, 1987) and reverse threading (Huber, T;Torda, A E, Protein Sci. 7(1): 142-149, 1998).

6. Hybridization

The oligonucleotide probe used in the characterization of the AmyS-likealpha-amylase above may suitably be prepared on the basis of the full orpartial nucleotide or amino acid sequence of the alpha-amylase inquestion.

Suitable conditions for assessing hybridization involve pre-soaking in5×SSC and pre-hybridizing for 1 hour at 40° C. in a solution of 20%formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50mg of denatured sonicated calf thymus DNA, followed by hybridization inthe same solution supplemented with 100 mM ATP for 18 hours at 40° C.,followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40°C. for 30 minutes (low stringency), preferred at 50° C. (mediumstringency), more preferably at 65° C. (high stringency), even morepreferably at 75° C. (very high stringency). More details about thehybridization method can be found in Sambrook et al., MOLECULAR CLONING:A LABORATORY MANUAL, 2^(nd) Ed., Cold Spring Harbor, 1989.

In the present context, “derived from” is intended not only to indicatean alpha-amylase produced or producible by a strain of the organism inquestion, but also an alpha-amylase encoded by a DNA sequence isolatedfrom such strain and produced in a host organism transformed with saidDNA sequence. Finally, the term is intended to indicate analpha-amylase, which is encoded by a DNA sequence of synthetic and/orcDNA origin and which has the identifying characteristics of thealpha-amylase in question. The term is also intended to indicate thatthe parent alpha-amylase may be a variant of a naturally occurringalpha-amylase, i.e., a variant, which is the result of a modification(insertion, substitution, deletion) of one or more amino acid residuesof the naturally occurring alpha-amylase.

7. General Mutations in Variant Alpha-Amylases

A variant described herein may, in one embodiment, comprise one or moremodifications in addition to those outlined above. Thus, it may beadvantageous that one or more proline residues (Pro) present in the partof the alpha-amylase variant that is modified is/are replaced with anon-proline residue which may be any of the possible, denaturant can beused to study equilibrium unfolding of the protein. This type ofspectroscopy can also be combined with devices, such as stopped flowmixers, to measure kinetics of protein folding/unfolding.

“Calcium dependent” means that, a particular enzyme requires calcium tosubstantially exhibit catalytic activity. Generally as used herein,“calcium dependent” encompasses a property of any enzyme that has astrict requirement for a divalent metal ion to exhibit catalyticactivity, and also includes enzymes whose catalytic activity issubstantially (e.g. more than 20%) increased in the presence of calciumor another divalent cation.

As used herein, “pH stable” with respect to an enzyme can refer to theenzyme activity or the protein conformation of the enzyme. In the firstsense, “pH stable” means the enzyme remains catalytically-active at aspecified pH or across a specified pH range. In the second sense, anenzyme may be deemed “stable” at a pH wherein the protein is notirreversibly denatured. In such a case, the enzyme would becomecatalytically active when returned to a pH capable of supportingcatalytic activity. pH stability may also be used in a relative orcomparative manner, for example, with a reference enzyme. In the presentapplication, an alpha-amylase variant can be more pH stable than awild-type B. licheniformis alpha-amylase when the variant has arelatively higher activity than the wild-type, e.g., when held at agiven pH or assayed under the same conditions, including pH. pH's ofmost interest are typically either the conditions of actual use, or pH'sthat are at or near the boundaries or extremes of the enzyme's naturalability to remain catalytically active.

“pH range” means a range of pH values e.g., from more acid to morebasic, or vice versa. With respect to an enzyme activity, a pH rangeindicates the upper and lower pH values at which the enzyme exhibits aspecified level of activity—e.g. a minimum activity, a specifiedpercentage of maximal activity, or a specified level of substrateconversion or product formation.

“Recombinant” when used in reference to a cell, nucleic acid, protein,or vector, indicates that the cell, nucleic acid, protein or vector, isthe result of, or has been modified by, the introduction of aheterologous sequence or the alteration of a native naturally occurringnon-proline residues, and which preferably is an alanine, glycine,serine, threonine, valine or leucine.

Analogously, in one embodiment, one or more cysteine residues present inthe parent alpha-amylase may be replaced with a non-cysteine residuesuch as serine, alanine, threonine, glycine, valine or leucine.

It is to be understood that the variants may incorporate two or more ofthe above outlined modifications.

Furthermore, it may be advantageous to introduce mutations in one ormore of the following positions (using SEQ ID NO: 7 for the numbering):

M15, V128, A111, H133, W138, T149, M197, N188, A209, A210, H405, T412,in particular the following single, double, triple, or multi mutations:

M15X, in particular M15T,L;

V128X, in particular V128E;

H133X, in particular H133Y;

N188X, in particular N188S,T,P;

M197X, in particular M197T,L;

A209X, in particular A209V;

M197T/W138F; M197T/138Y; M15T/H133Y/N188S;

M15N128E/H133Y/N188S; E119C/S130C; D124C/R127C; H133Y/T149I;

G475R, H133Y/S187D; H133Y/A209V.

In the case of the parent alpha-amylase having the amino acid sequenceshown in SEQ ID No. 7, relevant amino acid residues which may be deletedor substituted with a view to improving the oxidation stability includethe single cysteine residue (C363) and the methionine residues locatedin positions M8, M9, M96, M200, M206, M284, M307, M311, M316 and M438 inSEQ ID NO:2.

With respect to increasing the thermal stability of an alpha-amylasevariant relative to its parent alpha-amylase, it appears to beparticularly desirable to delete at least one, and preferably two, oreven three, of the following amino acid residues in the amino acidsequence shown in SEQ ID NO: 2: F178, R179, G180, I181, G182 and K183.

Particularly interesting pair-wise deletions of this type areR179*+G180*; and I181*+G182* (SEQ ID No. 16 or 15, respectively) (orequivalents of these pair-wise deletions in another alpha-amylasemeeting the requirements of a parent alpha-amylase in the context of thepresent disclosure).

Other residues of interest include N193F and V416G in the amino acidsequence shown in SEQ ID No. 2.

8. Altered Properties of Variants

8.1 General

The following section describes the relationship between mutations,which are present in a variant described herein, and desirablealterations in properties (relative to those of a parent AmyS-likealpha-amylase), which may result therefrom.

Described herein are AmyS-like alpha-amylases with altered properties.Parent alpha-amylases specifically contemplated herein are AmyS-likealpha-amylases and parent hybrid AmyS-like alpha-amylases.

In one embodiment, the Geobacillus stearothermophilus alpha-amylase (SEQID NO: 2) is used as the starting point, but in other embodiments, theSP722, BLA, BAN, AA560, SP690, KSM AP1378, #707 and other Bacillusalpha-amylases may be used. Amino acid positions corresponding topositions in SEQ ID NO: 2 are readily determined in accordance herewith.

The skilled artisan will appreciate that while any parent alpha-amylasecould be used as a reference amylase for the purpose ofnumbering/identifying the amino acid residues modified or to be modifiedin a particular variant, SEQ ID NO: 1 is presently a preferred sequencefor such purpose, because it is the longest B. stearothermophilussequence presently available herein.

In one aspect, this disclosure relates to variant with alteredproperties, e.g., as described above.

In one of its several aspects, this disclosure provides a variant of aparent G. stearothermophilus alpha-amylase, comprising an alteration atone or more positions (using e.g., SEQ ID NO: 1 for the amino acidnumbering) selected from the group of:

P17, D19, T21, N28, S51, G72, V74, A82, Q86, Q89, A93, G95, Q97, W115,D117, P123, S124, D125, N127, I130, G132, Q135, P145, G146, G148, S153,Y159, W166, S169, K171, W187, P209, N224, S242, G256, D269, N271, T278,N281, G302, A304, R308, T321, Q358, P378, S382, K383, T398, H405, T417,E418, P420, G421, P432, W437, G446, G454, S457, T459, T461, S464, G474,R483,

wherein

(a) the alteration(s) are independently (i) an insertion of an aminoacid downstream of the amino acid that occupies the position; (ii) adeletion of the amino acid that occupies the position; or (iii) asubstitution of the amino acid that occupies the position with adifferent amino acid,

(b) the variant has alpha-amylase activity, and

(c) each position corresponds to a position of the amino acid sequenceof the parent amylase, e.g., a G. stearothermophilus alpha-amylase,e.g., having the amino acid sequence shown in SEQ ID NO: 2, e.g., atruncated alpha-amylase that is available commercially as SPEZYME XTRAfrom Genencor.

Specifically contemplated herein are S242A, S242Q, S242N and S242Evariants.

Additionally, residues R179, G180, I181, G182, and K183 were chosen toexplore the effect of mutations in the calcium-sodium binding region,and P245 was chosen because a proline in the middle of an alpha-helix isunusual.

Corresponding positions in other parent AmyS-like alpha-amylases can befound by alignment as described above, for example, as with thosesequences shown in the alignment in FIG. 4. Thus, variants of a parentAmyS-like alpha-amylase, comprising an alteration at one or more of theabove enumerated positions (using, e.g., SEQ ID NO: 1 for comparativeamino acid numbering) is contemplated herein.

8.2 Altered Properties: Stability

In the context of the variants described herein, mutations (includingamino acid substitutions and deletion) of importance with respect toachieving altered stability, in particular improved stability (i.e.,higher or lower), at especially high temperatures (i.e., 70-120° C.)and/or extreme pH (i.e. low or high pH, i.e, pH 4-6 or pH 8-11,respectively), in particular at free (i.e., unbound, therefore insolution) calcium concentrations below 60 ppm, include any of themutations listed in the “Altered Properties” section. The stability maybe determined as described in the “Methods” section below.

8.3 Altered Properties: Ca²⁺ Stability

Altered Ca²⁺ stability means the stability of the enzyme under Ca²⁺depletion has been improved, i.e., higher or lower stability, relativeto the parent enzyme. In the context of the presently describedvariants, mutations (including amino acid substitutions and deletions)of importance with respect to achieving altered Ca²⁺ stability, inparticular improved Ca²⁺ stability, i.e., higher or lower stability, atespecially high pH (i.e., pH 8-10.5) include any of the mutations listedin the “Altered Properties” section.

8.4 Altered Properties: Specific Activity

In a further aspect, important mutations (including amino acidsubstitutions and deletions) with respect to obtaining variantsexhibiting altered specific activity, in particular increased ordecreased specific activity, especially at temperatures from 10-60° C.,preferably 20-50° C., especially 30-40° C., include any of the mutationslisted in the in “Altered properties” section. The specific activity maybe determined as described in the “Methods” section below.

8.5 Altered Properties: Oxidation Stability

The described variants may have altered oxidation stability, inparticular higher oxidation stability, in comparison to the parentalpha-amylase. Increased oxidation stability is advantageous in, e.g.,detergent compositions and decreased oxidation stability may beadvantageous in compositions intended for starch liquefaction. Oxidationstability may be determined as described in the “Methods” section below.

8.6 Altered Properties: Altered pH Profile

Important positions and mutations with respect to obtaining variantswith altered pH profile, in particular improved activity at especiallyhigh pH (i.e., pH 8-10.5) or low pH (i.e., pH 4-6) include mutations ofamino residues located close to the active site residues.

Preferred specific mutations/substitutions include those listed above inthe section “Altered Properties” for the positions in question. Suitableassays are described in the “Methods” section below.

8.7 Altered Properties: Wash Performance

Important positions and mutations with respect to obtaining variantswith improved wash performance at especially high pH (i.e., pH 8.5-11)include the specific mutations/substitutions listed above in the section“Altered Properties” for the positions in question. The wash performancemay be tested as described below in the “Methods” section.

9. Methods of Preparing α-Amylase Variants

Methods for introducing mutations into genes are known in the art, asare cloning methods for α-amylase-encoding DNA sequences. Such methodsincluding methods for generating mutations at specific sites within theα-amylase-encoding sequence will be discussed below.

9.1 Cloning a DNA Sequence Encoding an α-Amylase

The DNA sequence encoding a parent α-amylase may be isolated from anycell or microorganism producing the α-amylase in question, using variousmethods well known in the art. First, a genomic DNA and/or cDNA libraryshould be constructed using chromosomal DNA or messenger RNA from theorganism that produces the α-amylase to be studied. If the amino acidsequence of the α-amylase is known, homologous, labeled oligonucleotideprobes may be synthesized and used to identify α-amylase-encoding clonesfrom a genomic library prepared from the organism in question.Alternatively, a labeled oligonucleotide probe containing sequenceshomologous to a known α-amylase gene can be used as a probe to identifyα-amylase-encoding clones, e.g., using hybridization and washingconditions of lower stringency.

Another method for identifying α-amylase-encoding clones is based oninserting fragments of genomic DNA into an expression vector, such as aplasmid, transforming α-amylase-negative bacteria with the resultinggenomic DNA library, and plating the transformed bacteria onto agarcontaining a substrate for α-amylase, thereby allowing clones expressingthe α-amylase to be readily identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established, standard methods, e.g. the phosphoamiditemethod described by S. L. Beaucage and M. H. Caruthers, TetrahedronLetters 22: 1859-1869 (1981) or the method described by Matthes et al.,EMBO J. 3:801-895 (1984). In the phosphoamidite method, oligonucleotidesare synthesized, e.g., in an automatic DNA synthesizer, purified,annealed, ligated, and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed origin comprising e.g.,genomic and synthetic sequences, synthetic and cDNA sequences, orgenomic and cDNA sequences, prepared by ligating fragments of synthetic,genomic, or cDNA origin (as appropriate, the fragments corresponding tovarious parts of the entire DNA sequence), in accordance with standardtechniques. The DNA sequence may also be prepared by polymerase chainreaction (PCR) using specific primers, for instance as described in U.S.Pat. No. 4,683,202 or R. K. Saiki et al. EMBO J. 3:801-895 (1988).

9.2 Site-Directed Mutagenesis

Once an α-amylase-encoding DNA sequence has been isolated, and desirablesites for mutation identified, mutations may be introduced usingsynthetic oligonucleotides. These oligonucleotides contain nucleotidesequences flanking the desired mutation sites; mutant nucleotides areinserted during oligonucleotide synthesis. In a specific method, asingle-stranded gap of DNA, bridging the α-amylase-encoding sequence, iscreated in a vector carrying the α-amylase gene. Then the syntheticnucleotide, bearing the desired mutation, is annealed to a homologousportion of the single-stranded DNA. The remaining gap is then filled inwith DNA polymerase I (Klenow fragment) and the construct is ligatedusing T4 ligase. A specific example of this method is described inMorinaga et al. Biotechnology 2:636-639 (1984). U.S. Pat. No. 4,760,025discloses the introduction of oligonucleotides encoding multiplemutations by performing minor alterations of the cassette. However, aneven greater variety of mutations can be introduced at any one time bythe Morinaga method, because a multitude of oligonucleotides, of variouslengths, can be introduced.

Another method of introducing mutations into α-amylase-encoding DNAsequences is described in Nelson and Long, Analytical Biochem.,180:147-151 (1989). It involves the 3-step generation of a PCR fragmentcontaining the desired mutation introduced by using a chemicallysynthesized DNA strand as one of the primers in the PCR reactions. Fromthe PCR-generated fragment, a DNA fragment carrying the mutation may beisolated by cleavage with restriction endonucleases and reinserted intoan expression plasmid.

The skilled artisan will appreciate that many alternative methods areavailable for providing or obtaining variants herein. For example, geneshuffling, e.g., as described in WO 95/22625 (from Affymax TechnologiesN.V.) or in WO 96/00343 (from Novo Nordisk A/S), or other correspondingtechniques resulting in hybrid enzymes comprising the mutation(s), e.g.,substitution(s) and/or deletion(s), in question.

9.3 Expression of Alpha-Amylase Variants

A DNA sequence encoding the variant produced by methods described above,or by any alternative methods known in the art, can be expressed, inenzyme form, using an expression vector which typically includes controlsequences encoding a promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a repressor gene orvarious activator genes.

The recombinant expression vector carrying the DNA sequence encoding analpha-amylase variant for use herein may be any vector, which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e., a vector that exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, a bacteriophage, an extrachromosomal element, a minichromosome,or an artificial chromosome. Alternatively, the vector may be integratedinto the host cell genome and replicated together with the chromosome(s)into which it has been integrated.

In the vector, the DNA sequence should be operably-connected to asuitable promoter sequence. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding an alpha-amylase variant foruse herein, especially in a bacterial host, are the promoter of the lacoperon of E. coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis alpha-amylasegene (amyL), the promoters of the Geobacillus stearothermophilusmaltogenic amylase gene (amyM), the promoters of the Bacillusamyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillussubtilis xylA and xylB genes etc. For transcription in a fungal host,examples of useful promoters are those derived from the gene encoding A.oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. nigerneutral alpha-amylase, A. niger acid stable alpha-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase or A. nidulans acetamidase.

Expression vectors for use herein may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably-connected to the DNA sequence encoding the alpha-amylasevariant. Termination and polyadenylation sequences may suitably bederived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one that confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD, and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

While intracellular expression may be advantageous in some respects,e.g., when using certain bacteria as host cells, it is generallypreferred that the expression is extracellular. In general, the Bacillusalpha-amylases mentioned herein comprise a pre-region permittingsecretion of the expressed protease into the culture medium. Ifdesirable, this pre-region may be replaced by a different pre-region orsignal sequence, conveniently accomplished by substitution of the DNAsequences encoding the respective pre-regions.

The procedures used to ligate a DNA construct encoding an alpha-amylasevariant, the promoter, terminator and other elements, respectively, andto insert them into suitable vectors containing the informationnecessary for replication, are well known to persons skilled in the art(cf., for instance, Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2^(nd) Ed., Cold Spring Harbor, 1989).

Cells for use herein, e.g. comprising a DNA construct or an expressionvector as defined above, can be used as host cells in the recombinantproduction of an alpha-amylase variant. The cell may be transformed witha DNA construct encoding the variant, conveniently by integrating theDNA construct (in one or more copies) in the host chromosome. Thisintegration is generally considered to be an advantage as the DNAsequence is more likely to be stably maintained in the cell. Integrationof the DNA constructs into the host chromosome may be performedaccording to conventional methods, e.g., by homologous or heterologousrecombination. Alternatively, the cell may be transformed with anexpression vector as described above in connection with the differenttypes of host cells.

Cells for use herein may be cells of a higher organism such as a mammalor an insect, but are preferably microbial cells, e.g., a bacterial or afungal (including yeast) cell.

Examples of suitable bacteria are Gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Geobacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram-negative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

Where used for expression, a yeast may favorably be selected from aspecies of Saccharomyces or Schizosaccharomyces, e.g. Saccharomycescerevisiae. A filamentous fungus may advantageously be selected from aspecies of Aspergillus, e.g., Aspergillus oryzae or Aspergillus niger.Fungal cells may be transformed by a process involving protoplastformation and transformation of the protoplasts followed by regenerationof the cell wall in a manner known per se. A suitable procedure fortransformation of Aspergillus host cells is described in EP 238 023.

In a yet further aspect, the disclosure relates to a method of producingan alpha-amylase variant, which method comprises cultivating a host cellas described above under conditions conducive to the production of thevariant and recovering the variant from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the alpha-amylase variant. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g., as described in catalogues of the ATCC).

The alpha-amylase variant secreted from the host cells may be recoveredfrom the culture medium by known procedures, including separating thecells from the medium by centrifugation or filtration, and precipitatingproteinaceous components of the medium by means of a salt such asammonium sulphate, followed by the use of chromatographic proceduressuch as ion exchange chromatography, affinity chromatography, or thelike.

9.4 Methods for Characterizing and Screening Variants

9.4.1 Filter Screening Assays

The below assays may be used to screening of AmyS-like alpha-amylasevariants having altered stability at high or low pH and/or under Ca²⁺depleted conditions compared to the parent enzyme and AmyS-likealpha-amylase.

9.4.2 High pH Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67,Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plateswith 10 micro g/ml kanamycin at 37° C. for at least 21 hours. Thecellulose acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating,but before incubation in order to be able to localize positive variantson the filter and the nitrocellulose filter with bound variants istransferred to a container with glycine-NaOH buffer, pH 8.6-10.6 andincubated at room temperature (can be altered from 10-60° C.) for 15min. The cellulose acetate filters with colonies are stored on theTY-plates at room temperature until use. After incubation, residualactivity is detected on plates containing 1% agarose, 0.2% starch inglycine-NaOH buffer, pH 8.6-10.6. The assay plates with nitrocellulosefilters are marked the same way as the filter sandwich and incubated for2 hours at room temperature. After removal of the filters, the assayplates are stained with 10% Lugol solution. Starch degrading variantsare detected as white spots on dark blue background and then identifiedon the storage plates. Positive variants are rescreened twice under thesame conditions as the first screen.

9.4.3 Low Calcium Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67,Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plateswith a relevant antibiotic, e.g., kanamycin or chloramphenicol, at 37°C. for at least 21 hours. The cellulose-acetate layer is located on theTY agar plate.

Each filter sandwich is specifically marked with a needle after plating,but before incubation in order to be able to localize positive variantson the filter and the nitrocellulose filter with bound variants istransferred to a container with carbonate/bicarbonate buffer pH 8.5-10and with different EDTA concentrations (0.001 mM-100 mM). The filtersare incubated at room temperature for 1 hour. The cellulose acetatefilters with colonies are stored on the TY-plates at room temperatureuntil use. After incubation, residual activity is detected on platescontaining 1% agarose, 0.2% starch in carbonate/bicarbonate buffer pH8.5-10. The assay plates with nitrocellulose filters are marked the sameway as the filter sandwich and incubated for 2 hours at roomtemperature. After removal of the filters the assay plates are stainedwith 10% Lugol solution. Starch degrading variants are detected as whitespots on dark blue background and then identified on the storage plates.Positive variants are rescreened twice under the same conditions as thefirst screen.

9.4.4 Low pH Filter Assay

Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67,Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dasseli Germany) on TY agar plateswith 10 micro g/ml chloramphenicol at 37° C. for at least 21 hours. Thecellulose acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating,but before incubation in order to be able to localize positive variantson the filter, and the nitrocellulose filter with bound variants istransferred to a container with citrate buffer, pH 4.5 and incubated at80° C. for 20 minutes (when screening for variants in the wild-typebackbone) or at 85° C. for 60 minutes (when screening for variants ofthe parent alpha-amylase). The cellulose acetate filters with coloniesare stored on the TY-plates at room temperature until use. Afterincubation, residual activity is detected on assay plates containing 1%agarose, 0.2% starch in citrate buffer, pH 6.0. The assay plates withnitrocellulose filters are marked the same way as the filter sandwichand incubated for 2 hours at 50° C. After removal of the filters, theassay plates are stained with 10% Lugol solution. Starch degradingvariants are detected as white spots on dark blue background and thenidentified on the storage plates. Positive variants are re-screenedtwice under the same conditions as the first screen.

9.4.5 Secondary Screening

Positive transformants after rescreening are picked from the storageplate and tested in a secondary plate assay. Positive transformants aregrown for 22 hours at 37° C. in 5 mL LB+chloramphenicol. The Bacillusculture of each positive transformant and as a control a cloneexpressing the corresponding backbone are incubated in citrate buffer,pH 4.5 at 90° C. and samples are taken at 0, 10, 20, 30, 40, 60 and 80minutes. A 3 μL sample is spotted on an assay plate. The assay plate isstained with 10% Lugol solution. Improved variants are seen as variantswith higher residual activity (detected as halos on the assay plate)than the backbone. The improved variants are determined by nucleotidesequencing.

9.4.6 Stability Assay of Unpurified Variants

The stability of the variants may be assayed as follows: Bacilluscultures expressing the variants to be analyzed are grown for 21 hoursat 37° C. in 10 mLLB with chloramphenicol. 800 microliter culture ismixed with 200 microliter citrate buffer, pH 4.5. A number of 70 μLaliquots corresponding to the number of sample time points are made inPCR tubes and incubated at 70° C. or 90° C. for various time points(typically 5, 10, 15, 20, 25 and 30 minutes) in a PCR machine. The 0 minsample is not incubated at high temperature. Activity in the sample ismeasured by transferring 20 microliter to 200 microliter of thealpha-amylase PNP-G₇ substrate MPR3 ((Boehringer Mannheim Cat. No.1660730) as described below under “Assays for Alpha-Amylase Activity”.Results are plotted as percentage activity (relative to the 0 timepoint) versus time, or stated as percentage residual activity afterincubation for a certain period of time.

9.4.7 Fermentation and Purification of Alpha-Amylase Variants

A B. subtilis strain harboring the relevant expression plasmid may befermented and purified as follows: The strain is streaked on a LB-agarplate with 10 μg/ml kanamycin from −80° C. stock, and grown overnight at37° C. The colonies are transferred to 100 mL PS-1 media supplementedwith 10 μg/ml chloramphenicol in a 500 mL shaking flask.

Composition of PS-1 medium Pearl sugar 100 g/l Soy Bean Meal 40 g/lNa₂HPO₄, 12 H₂O 10 g/l Pluronic ™ PE 6100 0.1 g/l CaCO₃ 5 g/lThe culture is shaken at 37° C. at 270 rpm for 5 days.

Cells and cell debris are removed from the fermentation broth bycentrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatantis filtered to obtain a completely clear solution. The filtrate isconcentrated and washed on a UF-filter (10000 cut off membrane) and thebuffer is changed to 20 mM acetate pH 5.5. The UF-filtrate is applied onan S-SEPHAROSE F.F. and elution is carried out by step elution with 0.2MNaCl in the same buffer. The eluate is dialyzed against 10 mM Tris, pH9.0 and applied on a Q-SEPHAROSE F.F. and eluted with a linear gradientfrom 0-0.3M NaCl over 6 column volumes. The fractions that contain theactivity (measured by the PHADEBAS assay) are pooled, pH was adjusted topH 7.5 and remaining color was removed by treatment with 0.5% w/v activecharcoal in 5 minutes.

9.4.8 Specific Activity Determination

The specific activity is determined using the PHADEBAS® assay (MagleLife Sciences) as activity/mg enzyme. The manufactures instructions arefollowed (see also below under “Assay for Alpha-Amylase Activity).

9.4.9 Determination of Isoelectric Point

The pI is determined by isoelectric focusing (ex: Pharmacia, Ampholine,pH 3.5-9.3).

9.4.10 Stability Determination

The amylase stability may be measured using the method as follows:

The enzyme is incubated under the relevant conditions. Samples are takenat various time points, e.g., after 0, 5, 10, 15 and 30 minutes anddiluted 25 times (same dilution for all taken samples) in assay buffer(50 mM Britton buffer pH 7.3) and the activity is measured using thePHADEBAS assay (Magle Life Sciences) under standard conditions pH 7.3,37° C.

The activity measured before incubation (0 minutes) is used as reference(100%). The decline in percent is calculated as a function of theincubation time. The table shows the residual activity after, e.g., 30minutes of incubation.

9.4.11 Assays for Alpha-Amylase Activity

1. PHADEBAS Assay

Alpha-amylase activity is determined by a method employing PHADEBAS®tablets as substrate. PHADEBAS tablets (PHADEBAS® Amylase Test, suppliedby Magle Life Sciences) contain a cross-linked insoluble blue-coloredstarch polymer, which has been mixed with bovine serum albumin and abuffer substance and tablet.

For every single measurement one tablet is suspended in a tubecontaining 5 m L50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric add, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to the valueof interest with NaOH). The test is performed in a water bath at thetemperature of interest. The alpha-amylase to be tested is diluted in 50mM Britton-Robinson buffer. One milliliter of this alpha-amylasesolution is added to the 5 mL 50 mM Britton-Robinson buffer. The starchis hydrolyzed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

It is important that the measured 620 nm absorbance after 10 or 15minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range, there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions), 1 mg of a given alpha-amylase will hydrolyze a certainamount of substrate and a blue color will be produced. The colorintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

2. Alternative Method

Alpha-amylase activity is determined by a method employing the PNP-G₇substrate. PNP-G₇ which is a abbreviation forp-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide whichcan be cleaved by an endo-amylase. Following the cleavage, thealpha-Glucosidase included in the kit digest the substrate to liberate afree PNP molecule which has a yellow color and thus can be measured byvisible spectrophotometry at λ=405 nm (400-420 nm). Kits containingPNP-G₇ substrate and alpha-Glucosidase is manufactured byBoehringer-Mannheim (cat. No. 1054635).

To prepare the reagent solution 10 mL of substrate/buffer solution isadded to 50 mL enzyme/buffer solution as recommended by themanufacturer. The assay is performed by transferring 20 micro I sampleto a 96 well microtitre plate and incubating at 25° C. 200 μL reagentsolution pre-equilibrated to 25° C. is added. The solution is mixed, andpre-incubated 1 minute, and absorption is measured every 30 seconds over4 minutes at OD 405 mm in an ELISA reader.

The slope of the time dependent absorption-curve is directlyproportional to the activity of the alpha-amylase in question under thegiven set of conditions.

9.4.12 Determination of LAS Sensitivity

The variant is incubated with different concentrations of LAS (linearalkyl benzene sulphonate; Nansa 1169/P) for 10 minutes at 40° C.

The residual activity is determined using the PHADEBAS® assay method orthe alternative method employing the PNP-G₇ substrate.

LAS is diluted in 0.1 M phosphate buffer pH 7.5.

The following concentrations are used:

500 ppm, 250 ppm, 100 ppm, 50 ppm, 25 ppm, and 10 ppm on no LAS.

The variant is diluted in the different LAS buffers to concentration of0.01-5 mg/l in a total volume of 10 mL and incubated for 10 minutes in atemperature controlled water bath. The incubation is stopped bytransferring a small aliquot into cold assay buffer. It is importantthat during activity measurement the LAS concentration is below 1 ppm,in order not to affect the activity measurement. The residual activityis determined in duplicate using the above mentioned PHADEBAS® assay oralternative method. The activity is measured after subtraction of theblank. The activity with no LAS is 100%.

10. Methods of Using the Amylase Variants: Industrial Applications

The alpha-amylase variants presented herein possess valuable propertiesallowing for a variety of industrial applications. One or more of thevariant enzymes or compositions described herein may also be used indetergents, in particular laundry detergent compositions and dishwashingdetergent compositions, hard surface cleaning compositions, and incomposition for desizing of textiles, fabrics or garments, forproduction of pulp and paper, beer making, ethanol production, andstarch conversion processes as described above.

One or more of the variants with altered properties may be used forstarch processes, in particular starch conversion, especiallyliquefaction of starch (see, e.g., U.S. Pat. No. 3,912,590, EP PatentApplication Nos. 252,730 and 63,909, WO 99/19467, and WO 96/28567 allreferences hereby incorporated by reference). Also contemplated arecompositions for starch conversion purposes, which may also comprise aglucoamylase, pullulanase, and/or other alpha-amylase(s).

Further, one or more of the variants are also particularly useful in theproduction of sweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017hereby incorporated by reference), such as fuel, drinking and industrialethanol, from starch or whole grains.

The variants herein may also be useful for desizing of textiles,fabrics, and garments (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736,EP 119,920 hereby incorporated by reference), beer making or brewing,and in pulp and paper production or related processes.

10.1 Pre-Treatment of Native Starch

Native starch consists of microscopic granules, which are insoluble inwater at room temperature. When an aqueous starch slurry is heated, thegranules swell and eventually burst, dispersing the starch moleculesinto the solution. During this “gelatinization” process there is adramatic increase in viscosity. As the solids level is 30-40% in atypical industrial process, the starch has to be thinned or “liquefied”so that it can be suitably processed. This reduction in viscosity isprimarily attained by enzymatic degradation in current commercialpractice.

10.2 Starch Conversion

Conventional starch-conversion processes, such as liquefaction andsaccharification processes are described, e.g., in U.S. Pat. No.3,912,590 and EP Patent Publications Nos. 252,730 and 63,909, each ofwhich are hereby incorporated by reference herein.

In an embodiment, the conversion process degrading starch to lowermolecular weight carbohydrate components such as sugars or fat replacersincludes a debranching step.

10.3 Starch to Sugar Conversion

In the case of converting starch into a sugar, the starch isdepolymerized. Such a depolymerization process consists of, e.g., apre-treatment step and two or three consecutive process steps, viz aliquefaction process, a saccharification process, and depending on thedesired end-product, an optional isomerization process.

10.4 Isomerization

When the desired final sugar product is, e.g., high fructose syrup thedextrose syrup may be converted into fructose. After thesaccharification process, the pH is increased to a value in the range of6-8, preferably pH 7.5, and the calcium is removed by ion exchange. Thedextrose syrup is then converted into high fructose syrup using, e.g.,an immobilized glucose isomerase (such as Gensweet® IGI-HF).

10.5 Ethanol Production, Other Fermentation

In general, alcohol production (ethanol) from whole grain can beseparated into 4 main steps:

Milling

Liquefaction

Saccharification

Fermentation

10.6 Milling

The grain is milled in order to open up the structure and allow forfurther processing. Two processes used are wet or dry milling. In drymilling, the whole kernel is milled and used in the remaining part ofthe process. Wet milling gives a very good separation of germ and meal(starch granules and protein) and is with a few exceptions applied atlocations where there is a parallel production of syrups.

10.7 Liquefaction

In the liquefaction process the starch granules are solubilized byhydrolysis to maltodextrins mostly of a DP higher than 4. The hydrolysismay be carried out by acid treatment or enzymatically by alpha-amylase.Acid hydrolysis is used on a limited basis.

The raw material can be milled whole grain or a side stream from starchprocessing.

During a typical enzymatic liquefaction, the long-chained starch isdegraded into branched and linear shorter units (maltodextrins) by analpha-amylase. Enzymatic liquefaction is generally carried out as athree-step hot slurry process. The slurry is heated to between 60-95° C.(preferably 77-86° C., 80-85° C., and 83-85° C.) and the enzyme(s) is(are) added. The liquefaction process is carried out at 105-11° C. for 5to 10 minutes followed by 1-2 hours at 95° C. The pH is generallybetween 5.5 and 6.2. In order to ensure optimal enzyme stability underthese conditions, 1 mM of calcium is added (to provide about 40 ppm freecalcium ions). After such treatment, the liquefied starch will have a“dextrose equivalent” (DE) of 10-15.

The slurry is subsequently jet-cooked at between 95-140° C., preferably105-125° C., cooled to 60-95° C. and more enzyme(s) is (are) added toobtain the final hydrolysis. The liquefaction process is carried out atpH 4.5-6.5, typically at a pH between 5 and 6. Milled and liquefiedgrain is also known as mash.

10.8 Saccharification and Fermentation

Liquefied starch-containing material is saccharified in the presence ofsaccharifying enzymes such as glucoamylases. The saccharificationprocess may last for 12 hours to 120 hours (e.g. 12 to 90 hours, 12 to60 hours and 12 to 48 hours). However, it is common to perform apre-saccharification step for about 30 minutes to 2 hours (e.g., 30 to90 minutes) in a temperature range of 30 to 65° C. and typically around60° C. which is followed by a complete saccharification duringfermentation referred to as simultaneous saccharification andfermentation (SSF). The pH is usually between 4.2-4.8, preferably pH4.5. In a simultaneous saccharification and fermentation (SSF) process,there is no holding stage for saccharification, rather, the yeast andenzymes are added together.

In a typical saccharification process, maltodextrins produced duringliquefaction are converted into dextrose by addition of a glucoamylase(e.g., OPTIDEX® L-400) and a debranching enzyme, such as an isoamylase(U.S. Pat. No. 4,335,208) or a pullulanase. The temperature is loweredto 60° C., prior to addition of the glucoamylase and debranching enzymeare added. The saccharification process proceeds for 24-72 hours.

Prior to addition of the saccharifying enzymes, the pH is reduced tobelow 4.5, while maintaining a high temperature (above 95° C.), toinactivate the liquefying alpha-amylase. This process reduces theformation of short oligosaccharide called “panose precursors,” whichcannot be hydrolyzed properly by the debranching enzyme. Normally, about0.2-0.5% of the saccharification product is the branched trisaccharidepanose (Glc pα1-6Glc pα1-4Glc), which cannot be degraded by apullulanase. If active amylase from the liquefaction remains presentduring saccharification (i.e., no denaturing), the amount of panose canbe as high as 1-2%, which is highly undesirable since it lowers thesaccharification yield significantly.

Fermentable sugars, (e.g. dextrins, monosaccharides, particularlyglucose) are produced from enzymatic saccharification. These fermentablesugars may be further purified and/or converted to useful sugarproducts. In addition, the sugars may be used as a fermentationfeedstock in a microbial fermentation process for producingend-products, such as alcohol (e.g., ethanol and butanol), organic acids(e.g., succinic acid and lactic acid), sugar alcohols (e.g., glycerol),ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g.,lysine), proteins (e.g., antibodies and fragment thereof).

In a preferred embodiment, the fermentable sugars obtained during theliquefaction process steps are used to produce alcohol and particularlyethanol. In ethanol production, a SSF process is commonly used whereinthe saccharifying enzymes and fermenting organisms (e.g., yeast) areadded together and then carried out at a temperature of 30° C. to 40° C.

The organism used in fermentations will depend on the desiredend-product. Typically, if ethanol is the desired end product yeast willbe used as the fermenting organism. In some preferred embodiments, theethanol-producing microorganism is a yeast and specificallySaccharomyces such as strains of S. cerevisiae (U.S. Pat. No.4,316,956). A variety of S. cerevisiae are commercially available andthese include but are not limited to FALI (Fleischmann's Yeast),SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) andAngel alcohol yeast (Angel Yeast Company, China). The amount of starteryeast employed in the methods is an amount effective to produce acommercially significant amount of ethanol in a suitable amount of time,(e.g. to produce at least 10% ethanol from a substrate having between25-40% DS in less than 72 hours). Yeast cells are generally supplied inamounts of about 10⁴ to about 10¹², and preferably from about 10⁷ toabout 10¹⁰ viable yeast count per mL of fermentation broth. After yeastis added to the mash, it is typically subjected to fermentation forabout 24-96 hours, e.g., 35-60 hours. The temperature is between about26-34° C., typically at about 32° C., and the pH is from pH 3-6,preferably around pH 4-5.

The fermentation may include, in addition to a fermenting microorganisms(e.g. yeast), nutrients, and additional enzymes, including phytases. Theuse of yeast in fermentation is well known and reference is made to THEALCOHOL TEXTBOOK, K. JACQUES ET AL., EDS. 1999, NOTTINGHAM UNIVERSITYPRESS, UK.

In further embodiments, use of appropriate fermenting microorganisms, asis known in the art, can result in fermentation end product including,e.g., glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lacticacid, amino acids, and derivatives thereof. More specifically whenlactic acid is the desired end product, a Lactobacillus sp. (L. casei)may be used; when glycerol or 1,3-propanediol are the desiredend-products E. coli may be used; and when 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired endproducts, Pantoea citrea may be used as the fermenting microorganism.The above enumerated list are only examples and one skilled in the artwill be aware of a number of fermenting microorganisms that may be usedto obtain a desired end product.

10.9 Beer Making

The variant alpha-amylases provided for herein may also be very usefulin a beer-making process and similar fermentations; the alpha-amylaseswill typically be added during the mashing process. The process issubstantially similar to the milling, liquefaction, saccharification,and fermentation processes described above.

10.10 Using Amylase Variants for Starch Slurry Processing with Stillage

Milled starch-containing material is combined with water and recycledthin-stillage resulting in an aqueous slurry. The slurry can comprisebetween 15 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to40%, 20 to 35% and 30-36% ds). In some embodiments, the recycledthin-stillage (backset) is in the range of about 10 to 70% v/v (e.g., 10to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 60%, 20 to50%, 20 to 40% and also 20 to 30%).

Once the milled starch-containing material is combined with water andbackset, the pH is not adjusted in the slurry. Further the pH is notadjusted after the addition of phytase and optionally alpha-amylase tothe slurry. In a preferred embodiment, the pH of the slurry will be inthe range of about pH 4.5 to less than about 6.0 (e.g., pH 4.5 to 5.8,pH4.5 to 5.6, pH4.8 to 5.8, pH5.0 to 5.8, pH5.0 to 5.4, pH5.2 to 5.5 andpH5.2 to 5.9). The pH of the slurry may be between about pH 4.5 and 5.2depending on the amount of thin stillage added to the slurry and thetype of material comprising the thin stillage. For example, the pH ofthe thin stillage may be between pH 3.8 and pH 4.5. As a furtherexample, Table B below illustrates the pH change that occurs withaddition of increasing amounts of thin stillage to a whole ground cornslurry (32% ds) after stirring for 2 hours at 155° F.

TABLE B Thin stillage w/w % Final pH 0 5.52 20 5.29 40 5.16 50 5.09 605.05 80 4.98 100 4.94

During ethanol production, acids can be added to lower the pH in thebeer well, to reduce the risk of microbial contamination prior todistillation.

In some embodiments, phytase is added to the slurry. In otherembodiments, in addition to phytase, alpha-amylase is added to theslurry. In some embodiments, phytase and alpha-amylase are added to theslurry sequentially. In other embodiments, phytase and alpha-amylase areadded simultaneously. In some embodiments, the slurry comprising phytaseand optionally, alpha-amylase, are incubated (pretreated) for a periodof about 5 minutes to about 8 hours (e.g., 5 minutes to 6 hours, 5minutes to 4 hours, 5 minutes to 2 hours, and 15 minutes to 4 hours). Inother embodiments, the slurry is incubated at a temperature in the rangeof about 40 to 115° C., (e.g. 45 to 80° C., 50 to 70° C., 50 to 75° C.,60 to 110° C., 60 to 95° C., 70 to 110° C., 70 to 85° C. and 77 to 86°C.).

In other embodiments, the slurry is incubated at a temperature of about0 to about 30° C. (e.g. 0 to 25° C., 0 to 20° C., 0 to 15° C., 0 to 10°C. and 0 to 5° C.) below the starch gelatinization temperature of thestarch-containing material. In some embodiments, the temperature isbelow about 68° C., below about 65° C., below about 62° C., below about60° C. and below about 55° C. In some embodiments, the temperature isabove about 45° C., above about 50° C., above about 55° C. and aboveabout 60° C. In some embodiments, the incubation of the slurrycomprising a phytase and an alpha-amylase at a temperature below thestarch gelatinization temperature is referred to as a primary (1°)liquefaction.

In one embodiment, the milled starch-containing material is corn ormilo. The slurry comprises 25 to 40% ds, the pH is in the range of 4.8to 5.2, and the slurry is incubated with a phytase and optionally analpha-amylase for 5 minutes to 2 hours, at a temperature range of 60 to75° C.

Currently, it is believed that commercially-available microbialalpha-amylases used in the liquefaction process are generally not stableenough to produce liquefied starch substrate from a dry mill processusing whole ground grain at a temperature above about 80° C. at a pHlevel that is less than pH 5.6. The stability of many commerciallyavailable alpha-amylases is reduced at a pH of less than about 4.0.

In a further liquefaction step, the incubated or pretreatedstarch-containing material is exposed to an increase in temperature suchas about 0 to about 45° C. above the starch gelatinization temperatureof the starch-containing material (e.g. 70° C. to 120° C., 70° C. to110° C., and 70° C. to 90° C.) for a period of time of about 2 minutesto about 6 hours (e.g. 2 minutes to 4 hrs, 90 minutes, 140 minutes and90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferably between 1hour to 2 hours. The temperature can be increased by a conventional hightemperature jet cooking system for a short period of time, for example,for 1 to 15 minutes. Then the starch maybe further hydrolyzed at atemperature ranging from about 75° C. to 95° C., (e.g., 80° C. to 90° C.and 80° C. to 85° C.) for a period of about 15 to 150 minutes (e.g., 30to 120 minutes). In a preferred embodiment, the pH is not adjustedduring these process steps and the pH of the liquefied mash is in therange of about pH 4.0 to pH 5.8 (e.g., pH 4.5 to 5.8, pH 4.8 to 5.4, andpH 5.0 to 5.2). In some embodiments, a second dose of thermostablealpha-amylase is added to the secondary liquefaction step, but in otherembodiments there is no additional dosage of alpha-amylase.

The incubation and liquefaction steps may be followed bysaccharification and fermentation steps well known in the art.

10.11 Distillation

Optionally, following fermentation, alcohol (e.g., ethanol) may beextracted by, for example, distillation and optionally followed by oneor more process steps.

In some embodiments, the yield of ethanol produced by the methodsprovided herein is at least 8%, at least 10%, at least 12%, at least14%, at least 15%, at least 16%, at least 17% and at least 18% (v/v) andat least 23% v/v. The ethanol obtained according to the process providedherein may be used as, for example, fuel ethanol, drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

10.12 By-Products

Left over from the fermentation is the grain, which is typically usedfor animal feed either in liquid or dried form. In further embodiments,the end product may include the fermentation co-products such asdistiller's dried grains (DDG) and distiller's dried grain plus solubles(DDGS), which may be used, for example, as an animal feed.

Further details on how to carry out liquefaction, saccharification,fermentation, distillation, and recovery of ethanol are well known tothe skilled person.

According to the process provided herein, the saccharification andfermentation may be carried out simultaneously or separately.

10.13 Pulp and Paper Production

The variant alkaline alpha-amylase may also be used in the production oflignocellulosic materials, such as pulp, paper and cardboard, fromstarch reinforced waste paper and cardboard, especially where re-pulpingoccurs at pH above 7 and where amylases facilitate the disintegration ofthe waste material through degradation of the reinforcing starch. Thealpha-amylase variants are especially useful in a process for producinga papermaking pulp from starch-coated printed-paper. The process may beperformed as described in WO 95/14807, comprising the following steps:

a) disintegrating the paper to produce a pulp,

b) treating with a starch-degrading enzyme before, during or after stepa), and

c) separating ink particles from the pulp after steps a) and b).

The alpha-amylases may also be very useful in modifying starch whereenzymatically modified starch is used in papermaking together withalkaline fillers such as calcium carbonate, kaolin and clays. With thealkaline alpha-amylase variants it is possible to modify the starch inthe presence of the filler thus allowing for a simpler integratedprocess.

10.14 Desizing of Textiles, Fabrics and Garments

An alpha-amylase variant may also be very useful in textile, fabric orgarment desizing. In the textile processing industry, alpha-amylases aretraditionally used as auxiliaries in the desizing process to facilitatethe removal of starch-containing size, which has served as a protectivecoating on weft yams during weaving. Complete removal of the sizecoating after weaving is important to ensure optimum results in thesubsequent processes, in which the fabric is scoured, bleached and dyed.Enzymatic starch breakdown is preferred because it does not involve anyharmful effect on the fiber material. In order to reduce processing costand increase mill throughput, the desizing processing is sometimescombined with the scouring and bleaching steps. In such cases,non-enzymatic auxiliaries such as alkali or oxidation agents aretypically used to break down the starch, because traditionalalpha-amylases are not very compatible with high pH levels and bleachingagents. The non-enzymatic breakdown of the starch size does lead to somefiber damage because of the rather aggressive chemicals used.Accordingly, it would be desirable to use the alpha-amylase variants asthey have an improved performance in alkaline solutions. Thealpha-amylases may be used alone or in combination with a cellulase whendesizing cellulose-containing fabric or textile.

Desizing and bleaching processes are well known in the art. Forinstance, such processes are described in e.g., WO 95/21247, U.S. Pat.No. 4,643,736, EP 119,920 hereby incorporated by reference.

Commercially available products for desizing include OPTISIZE® FLEX fromGenencor.

10.15 Cleaning Processes and Detergent Compositions

The variant alpha-amylases described herein may be added to and thusbecome a component of a detergent composition for various cleaning orwashing processes, including laundry and dishwashing.

The detergent composition provided for herein may for example beformulated as a hand or machine laundry detergent composition, includinga laundry additive composition suitable for pretreatment of stainedfabrics and a rinse added fabric softener composition or be formulatedas a detergent composition for use in general household hard surfacecleaning operations, or be formulated for hand or machine dishwashingoperations.

In a specific aspect, there is provided for herein a detergent additivecomprising a variant enzyme described herein. The detergent additive aswell as the detergent composition may comprise one or more other enzymessuch as a protease, a lipase, a peroxidase, another amylolytic enzyme,e.g., another alpha-amylase, glucoamylase, maltogenic amylase, CGTaseand/or a cellulase mannanase (such as MANNASTAR™ from Danisco US Inc.,Genencor Division)), pectinase, pectin lyase, cutinase, and/or laccase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metalloprotease, preferably an alkaline microbial proteaseor a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likepro-teases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Preferred commercially available protease enzymes include ALCALASE®,SAVINASE®, PRIMASE®, DURALASE®, ESPERASE®, and KANNASE® (from NovozymesA/S), MAXATASE®, MAXACAL, MAXAPEM®, PROPERASE®, PURAFECT®, PURAFECTOXP®, FN2®, FN3®, FN4® (Genencor International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas spp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta,1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422). Other examples are lipase variants such as those described inWO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO97/04079 and WO 97/07202.

Preferred commercially available lipase enzymes include LIPOLASE™ andLIPOLASE ULTRA™ (Novozymes A/S).

Amylases: One or more additional amylases may also be included. Suitableamylases (alpha and/or beta) include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Amylases include, for example, alpha-amylases obtained from Bacillus,e.g., a special strain of B. licheniformis, described in more detail inGB 1,296,839. Examples of useful alpha-amylases are the variantsdescribed in WO 94/18314, WO 96/39528, WO 94/02597, WO 94/18314, WO96/23873, and WO 97/43424, especially the variants with substitutions inone or more of the following positions: 15, 23, 105, 106, 124, 128, 133,154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391,408, and 444.

Commercially available alpha-amylases are DURAMYL™, LIQUEZYME™ TERMAMY™,NATALASE™, FUNGAMYL™ and BAN™ (Novozymes A/S), RAPIDASE™ and PURASTAR™(from Genencor).

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Trichoderma, Humicola, Fusarium, Thielavia, Acremonium,e.g., the fungal cellulases produced from Humicola insolens,Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat.No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,776,757 and WO 89/09259. The Trichoderma reesei cellulasesare disclosed in U.S. Pat. No. 4,689,297, U.S. Pat. No. 5,814,501, U.S.Pat. No. 5,324,649, WO 92/06221 and WO 92/06165. Bacillus cellulases aredisclosed in U.S. Pat. No. 6,562,612.

Commercially available cellulases include CELLUZYME®, and CAREZYME®(Novozymes A/S), CLAZINASE®, and PURADAX HA® (Genencor InternationalInc.), and KAC-500(B)® (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include GUARDZYME® (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,e.g., a separate additive or a combined additive, can be formulated,e.g., granulate, a liquid, a slurry, etc. Preferred detergent additiveformulations are granulates, in particular non-dusting granulates,liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition may be in any convenient form, e.g., a bar, atablet, a powder, a granule, a paste or a liquid. A liquid detergent maybe aqueous, typically containing up to about 70% water and 0 to about30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of fromabout 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide,ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0 to about 65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system maycomprise peroxy acids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition may be stabilized usingconventional stabilizing agents, e.g., a polyol such as propylene glycolor glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or aboric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid, and thecomposition may be formulated as described in, e.g., WO 92/19709 and WO92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilre-deposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular one or more of the variant enzymes describedherein, may be added in an amount corresponding to 0.01-100 mg of enzymeprotein per liter of wash liquor, preferably 0.055 mg of enzyme proteinper liter of wash liquor, in particular 0.1-1 mg of enzyme protein perliter of wash liquor.

One or more of the variant enzymes described herein may additionally beincorporated in the detergent formulations disclosed in WO 97/07202,which is hereby incorporated as reference.

10.16 Dishwashing Detergent Compositions

The enzymes may also be used in dish wash detergent compositions,including the following:

1) Powder Automatic Dishwashing Composition

Nonionic surfactant 0.4-2.5% Sodium metasilicate  0-20% Sodiumdisilicate  3-20% Sodium triphosphate 20-40% Sodium carbonate  0-20%Sodium perborate 2-9% Tetraacetyl ethylene diamine (TAED) 1-4% Sodiumsulphate  5-33% Enzymes 0.0001-0.1%  2) Powder Automatic Dishwashing Composition

Nonionic surfactant 1-2%  (e.g. alcohol ethoxylate) Sodium disilicate2-30% Sodium carbonate 10-50%  Sodium phosphonate 0-5%  Trisodiumcitrate dihydrate 9-30% Nitrilotrisodium acetate (NTA) 0-20% Sodiumperborate monohydrate 5-10% Tetraacetyl ethylene diamine (TAED) 1-2% Polyacrylate polymer 6-25% (e.g. maleic acid/acrylic acid copolymer)Enzymes 0.0001-0.1%   Perfume 0.1-0.5%  Water 5-10%3) Powder Automatic Dishwashing Composition

Nonionic surfactant 0.5-2.0% Sodium disilicate 25-40% Sodium citrate30-55% Sodium carbonate  0-29% Sodium bicarbonate  0-20% Sodiumperborate monohydrate  0-15% Tetraacetyl ethylene diamine (TAED) 0-6%Maleic acid/acrylic 0-5% acid copolymer Clay 1-3% Polyamino acids  0-20%Sodium polyacrylate 0-8% Enzymes 0.0001-0.1%  4) Powder Automatic Dishwashing Composition

Nonionic surfactant 1-2% Zeolite MAP 15-42% Sodium disilicate 30-34%Sodium citrate  0-12% Sodium carbonate  0-20% Sodium perboratemonohydrate  7-15% Tetraacetyl ethylene 0-3% diamine (TAED) Polymer 0-4%Maleic acid/acrylic acid copolymer 0-5% Organic phosphonate 0-4% Clay1-2% Enzymes 0.0001-0.1%   Sodium sulphate Balance5) Powder Automatic Dishwashing Composition

Nonionic surfactant 1-7% Sodium disilicate 18-30% Trisodium citrate10-24% Sodium carbonate 12-20% Monopersulphate 15-21%(2KHSO₅•KHSO₄•K₂SO₄) Bleach stabilizer 0.1-2%   Maleic acid/acrylic acidcopolymer 0-6% Diethylene triamine pentaacetate,   0-2.5% pentasodiumsalt Enzymes 0.0001-0.1%   Sodium sulphate, water Balance6) Powder and Liquid Dishwashing Composition with Cleaning SurfactantSystem

Nonionic surfactant   0-1.5% Octadecyl dimethylamine N-oxide dihydrate0-5% 80:20 wt. C18/C16 blend of octadecyl dimethylamine 0-4% N-oxidedihydrate and hexadecyldimethyl amine N- oxide dihydrate 70:30 wt.C18/C16 blend of octadecyl bis 0-5% (hydroxyethyl)amine N-oxideanhydrous and hexadecyl bis (hydroxyethyl)amine N-oxide anhydrousC₁₃-C₁₅ alkyl ethoxysulfate with an average degree of  0-10%ethoxylation of 3 C₁₂-C₁₅ alkyl ethoxysulfate with an average degree of0-5% ethoxylation of 3 C₁₃-C₁₅ ethoxylated alcohol with an averagedegree of 0-5% ethoxylation of 12 A blend of C₁₂-C₁₅ ethoxylatedalcohols with an   0-6.5% average degree of ethoxylation of 9 A blend ofC₁₃-C₁₅ ethoxylated alcohols with an 0-4% average degree of ethoxylationof 30 Sodium disilicate  0-33% Sodium tripolyphosphate  0-46% Sodiumcitrate  0-28% Citric acid  0-29% Sodium carbonate  0-20% Sodiumperborate monohydrate   0-11.5% Tetraacetyl ethylene diamine (TAED) 0-4%Maleic acid/acrylic acid copolymer   0-7.5% Sodium sulphate   0-12.5%Enzymes 0.0001-0.1%  7) Non-Aqueous Liquid Automatic Dishwashing Composition

Liquid nonionic surfactant (e.g. alcohol ethoxylates)  2.0-10.0% Alkalimetal silicate  3.0-15.0% Alkali metal phosphate 20.0-40.0% Liquidcarrier selected from higher 25.0-45.0% glycols, polyglycols,polyoxides, glycolethers Stabilizer (e.g. a partial ester of phosphoricacid and a 0.5-7.0% C₁₆-C₁₈ alkanol) Foam suppressor (e.g. silicone)  0-1.5% Enzymes 0.0001-0.1%  8) Non-Aqueous Liquid Dishwashing Composition

Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0-10.0% Sodiumsilicate 3.0-15.0% Alkali metal carbonate 7.0-20.0% Sodium citrate0.0-1.5%  Stabilizing system (e.g. mixtures of finely divided 0.5-7.0% silicone and low molecular weight dialkyl polyglycol ethers) Lowmolecule weight polyacrylate polymer 5.0-15.0% Clay gel thickener (e.g.bentonite) 0.0-10.0% Hydroxypropyl cellulose polymer 0.0-0.6%  Enzymes0.0001-0.1%   Liquid carrier selected from higher lycols, polyglycols,Balance polyoxides and glycol ethers9) Thixotropic Liquid Automatic Dishwashing Composition

C₁₂-C₁₄ fatty acid  0-0.5% Block co-polymer surfactant 1.5-15.0% Sodiumcitrate 0-12% Sodium tripolyphosphate 0-15% Sodium carbonate 0-8% Aluminum tristearate  0-0.1% Sodium cumene sulphonate  0-1.7%Polyacrylate thickener 1.32-2.5%  Sodium polyacrylate 2.4-6.0%  Boricacid  0-4.0% Sodium formate   0-0.45% Calcium formate  0-0.2% Sodiumn-decydiphenyl oxide disulphonate  0-4.0% Monoethanol amine (MEA)  0-1.86% Sodium hydroxide (50%) 1.9-9.3%  1,2-Propanediol  0-9.4%Enzymes 0.0001-0.1%   Suds suppressor, dye, perfumes, water Balance10) Liquid Automatic Dishwashing Composition

Alcohol ethoxylate 0-20% Fatty acid ester sulphonate 0-30% Sodiumdodecyl sulphate 0-20% Alkyl polyglycoside 0-21% Oleic acid 0-10% Sodiumdisilicate monohydrate 18-33%  Sodium citrate dihydrate 18-33%  Sodiumstearate  0-2.5% Sodium perborate monohydrate 0-13% Tetraacetyl ethylenediamine (TAED) 0-8%  Maleic acid/acrylic acid copolymer 4-8%  Enzymes0.0001-0.1%  11) Liquid Automatic Dishwashing Composition Containing Protected BleachParticles

Sodium silicate  5-10% Tetrapotassium pyrophosphate 15-25% Sodiumtriphosphate 0-2% Potassium carbonate 4-8% Protected bleach particles,e.g. chlorine  5-10% Polymeric thickener 0.7-1.5% Potassium hydroxide0-2% Enzymes 0.0001-0.1%   Water Balance12) Automatic dishwashing compositions as described in 1), 2), 3), 4),6) and 10), wherein perborate is replaced by percarbonate.13) Automatic dishwashing compositions as described in 1)-6) whichadditionally contain a manganese catalyst. The manganese catalyst may,e.g., be one of the compounds described in “Efficient manganesecatalysts for low-temperature bleaching”, Nature 369: 637-39 (1994).14) Premium HDL Liquid Detergent Formulations

Bio-Soft S-101 Linear alkylbenzene sulfonic acid Steol CS-330 SodiumLaureth sulfate Bio-soft N25-7 Linear alkylethoxylate with 7 moles of EOStepanate SXS Sodium xylene sulfonate15) Ultra Liquid Detergent Formulation

Tionopal CBS-X Fluorescent whitening agent Alpha-step MC-48 Sodiumalpha-sulfomethylester Makon TD-6 Tridecylalcoholethoxylate

10.17 Use of Variants in Conjunction with Other Enzymes: Phytases

10.17.1 Use of Phytases

Phytases useful herein include enzymes capable of hydrolyzing phytatesand/or phytic acid under the conditions of use, e.g., the incubationand/or liquefaction steps. In some embodiments, the phytase is capableof liberating at least one inorganic phosphate from an inositolhexaphosphate (e.g., phytic acid). Phytases can be grouped according totheir preference for a specific position of the phosphate ester group onthe phytate molecule at which hydrolysis is initiated, (e.g., as3-phytases (EC 3.1.3.8) or as 6-phytases (EC 3.1.3.26)). A typicalexample of phytase is myo-inositol-hexakiphosphate-3-phosphohydrolase.

Phytases can be obtained from microorganisms such as fungal andbacterial organisms. Some of these microorganisms include e.g.Aspergillus (e.g., A. niger, A. terreus, A. ficum and A. fumigatus),Myceliophthora (M. thermophila), Talaromyces (T. thermophilus),Trichoderma spp (T. reesei), and Thermomyces (WO 99/49740). Alsophytases are available from Penicillium species, e.g., P. hordei (ATCCNo. 22053), P. piceum (ATCC No. 10519), or P. brevi-compactum (ATCC No.48944). See, for example U.S. Pat. No. 6,475,762. In addition, phytasesare available from Bacillus (e.g. B. subtilis, Pseudomonas, Peniophora,E. coli, Citrobacter, Enterbacter, and Buttiauxella (see e.g.WO2006/043178).

Commercial phytases are available such as NATUPHOS (BASF), RONOZYME P(Novozymes A/S), PHZYME (Danisco A/S, Diversa) and FINASE (AB Enzymes).The method for determining microbial phytase activity and the definitionof a phytase unit has been published by Engelen et al. (1994) J. of AOACInternational, 77: 760-764. The phytase may be a wild-type phytase, anactive variant or active fragment thereof.

In one embodiment, the phytase is derived from the bacteriumButtiauxiella spp. The Buttiauxiella spp. includes B. agrestis, B.brennerae, B. ferragutiase, B. gaviniae, B. izardii, B. noackiae, and B.warmboldiae. Strains of Buttiauxella species are available from DSMZ,the German National Resource Center for Biological Material(Inhoffenstrabe 7B, 38124 Braunschweig, Germany). Buttiauxella sp.strain P1-29 deposited under accession number NCIMB 41248 is an exampleof a particularly useful strain from which a useful phytase may beobtained. In some embodiments, the phytase is BP-wild-type, a variantthereof (such as BP-11) disclosed in WO 06/043178, or a variant asdisclosed in U.S. patent application Ser. No. 11/714,487, filed Mar. 6,2007. For example, a BP-wild-type and variants thereof are disclosed inTable 1 of WO 06/043178, wherein the numbering is in reference to SEQ IDNO: 3 of the published PCT application.

In one preferred embodiment, a useful phytase is one having at least75%, at least 80%, at least 85%, at least 88%, at least 90%, at least93%, at least 95%, at least 96%, at least 97%, at least 98% and at least99% sequence identity to the amino acid sequence set forth in SEQ ID NO:31 shown below or an active variant thereof. More preferably, thephytase will have at least 95% to 99% sequence identity with the aminoacid sequence set forth in SEQ ID NO: 31 or an active variant thereof.In some embodiments, the phytase comprises the amino acid sequence ofSEQ ID NO: 31. In yet others, the phytase is SEQ ID NO: 31.

Mature protein sequence of Buttiauxella BP-17 phytase (SEQ ID NO: 31)

NDTPASGYQV EKVVILSRHG VRAPTKMTQT MRDVTPNTWP EWPVKLGYIT PRGEHLISLMGGFYRQKFQQ QGILSQGSCP TPNSIYVWAD VDQRTLKTGE AFLAGLAPQC GLTIHHQQNLEKADPLFHPV KAGTCSMDKT QVQQAVEKEA QTPIDNLNQH YIPFLALMNT TLNFSTSAWCQKHSADKSCD LGLSMPSKLS IKDNGNKVAL DGAIGLSSTL AEIFLLEYAQ GMPQAAWGNIHSEQEWASLL KLHNVQFDLM ARTPYIARHN GTPLLQAISN ALNPNATESK LPDISPDNKILFIAGHDTNI ANIAGMLNMR WTLPGQPDNT PPGGALVFER LADKSGKQYV SVSMVYQTLEQLRSQTPLSL NQPAGSVQLK IPGCNDQTAE GYCPLSTFTR VVSQSVEPGC QLQ

In some embodiments, the amount (dosage) of phytase used in theincubation and/or liquefaction processes is in the range of about 0.001to 50 FTU/g ds, (e.g., in the range of about 0.01 to 25 FTU/g ds, about0.01 to 15 FTU/g ds, about 0.01 to 10 FTU/g ds, about 0.05 to 15 FTU/gds, or about 0.05 to 5.0 FTU/g).

10.17.2 Determination of Phytase Activity (FTU)

“Phytase Activity” (“FTU”) is measured by the release of inorganicphosphate. The inorganic phosphate forms a yellow complex with acidicmolybdate/vanadate reagent; and the yellow complex is measured at awavelength of 415 nm in a spectrophotometer and the released inorganicphosphate is quantified with a phosphate standard curve. One unit ofphytase (FTU) is the amount of enzyme that releases 1 micromole ofinorganic phosphate from phytate per minute under the reactionconditions given in the European Standard (CEN/TC 327,2005-TC327WI003270XX).

10.17.3 Determination of Phytic Acid Content

Phytic acid content: Phytic acid was extracted from the sample byadjusting the pH of the 5% slurry (if it is dry sample) to pH 10 andthen determined by an HPLC method using an ion exchange column. Phyticacid was eluted from the column using a NaOH gradient system. Phyticacid content in the liquid was then calculated by comparing to a phyticacid standard.

11. Compositions (Blends) Comprising the Variant Alpha-Amylases

In one of its several aspects, this disclosure provides compositionscomprising:

a) at least one alpha-amylase variant comprising an amino acid sequenceat least 95% identical to that of a parent AmyS-like alpha-amylase, andhaving a substitution at an amino acid position corresponding toposition 242 of a reference alpha-amylase, said variant havingdetectable alpha-amylase activity, and

b) at least one additional enzyme.

The variant is altered, as compared to a parent AmyS-like alpha-amylaseor a reference alpha-amylase, in one or more of any useful or measurableattributes including net charge, substrate specificity, substratecleavage, substrate binding, thermal stability, activity at one or morepH's, stability at one or more pH's, stability in oxidizing conditions,Ca²⁺ requirements, specific activity, catalytic rate, catalyticefficiency, activity in the presence of a phytate, thermal or pHstability in the presence of a phytate, ability to effect peak viscosityin a liquefaction test, or ability to effect final viscosity in aliquefaction test. In preferred embodiments, the variant will have morethan one altered attribute, for example, improved thermostability, andthe ability to reduce peak viscosity in a liquefaction, or the abilityto reduce both peak viscosity and final viscosity in a liquefaction, ascompared to a parent alpha-amylase, e.g. an AmyS-like amylase.

In various embodiments, the reference amylase is SEQ ID NO: 1 or 2.Other alpha-amylases can be used as the reference alpha-amylase. It ispreferred that the reference alpha-amylase for use herein have a serineresidue at amino acid position 242.

In various embodiments, the additional enzyme is a phytase, protease,lipase, pullulanase, glucoamylases, isomerase, or other enzymes usefulin a commercial process in conjunction with an alpha-amylase. Suchenzymes are known in the art in starch processing, sugar conversion,fermentations for alcohol and other useful end-products, commercialdetergents and cleaning aids, stain removal, fabric treatment ordesizing, and the like. Presently preferred additional enzymes arephytases. One embodiment uses a phytase that comprises SEQ ID NO: 17.

In certain embodiments, the variant is a S242A, S242D, S242E, S242F,S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant. Suchvariants are exemplified and characterized in the working examplesprovided herein.

The variant in some embodiments further comprises a sequencemodification at one or more amino acid positions corresponding to aminoacid positions 97, 179, 180, 193, 319, 349, 358, 416, 428, or 443 of thereference amylase, e.g., SEQ ID NO: 1 or 2. More specifically, thevariant comprises one or more of substitution at positions as follows: acysteine at 349, a cysteine at 428, a glutamic acid at 97, an arginineat 97, a glutamic acid at 319, an arginine at 319, a glutamic acid at358, an arginine at 358, a glutamic acid at 443, or an arginine at 443.Substitution of an N193 or a V416 or both, such as a substitution ofN193F or V416G, or both are useful herein for variants. Deletion ofamino acids corresponding to positions 179 and 180 may also be usedherein with any variant amylase.

In one embodiment of the composition, the alpha-amylase variant has atleast 95% homology to SEQ ID NO: 2 and comprises a substitution of aminoacid 242 relative to numbering in a reference amylase comprising theamino acid sequence SEQ ID NO: 1. As above, the variant preferably hasdetectable alpha-amylase activity under conditions permissive of suchactivity.

Useful parent amylases are discussed above. In some embodiments, theparent alpha-amylase is SEQ ID NO: 1, 2, 15, or 16. In others, theparent alpha-amylase is SEQ ID NO: 6, 7, 8, 9, 10, 11, or 12.

In various embodiments, the variant amylase and the phytase are presentin amounts such that the ratio of AAU:FTU is about 1:15 to about 15:1.Preferably, in some embodiments, the variant amylase and the phytase arepresent in amounts such that the ratio of AAU:FTU is about 1:4 to about3:1.

12. Methods for Using Variants with Other Enzymes

In another aspect, this disclosure provides methods of using the variantalpha-amylases in conjunction with other enzymes, particularly phytases.In one embodiment, methods are provided for treating a starch slurry.The treatment can be part of a liquefaction process, a saccharification,a fermentation process, and the like. The method generally comprises thesteps of

a) adding at least one phytase and at least one alpha-amylase to thestarch slurry, and b) incubating the starch slurry under conditionspermissive of activity of the phytase and the alpha-amylase. The methodencompasses any one or more adding steps such that the phytase and thealpha-amylase are added at, or about, the same time, or separately, inany order (i.e. phytase first or amylase first), with any useful amountof temporal separation between such adding steps. As with thecompositions, the alpha-amylase is a variant amylase comprising an aminoacid sequence at least 95% identical to that of a parent AmyS-likealpha-amylase, and having a substitution at an amino acid positioncorresponding to position 242 of a reference alpha-amylase. The varianthas detectable alpha-amylase activity.

In one embodiment, the variant is altered, as compared to a parentAmyS-like alpha-amylase or a reference amylase, in any one or more ofnet charge, substrate specificity, substrate cleavage, substratebinding, thermal stability, activity at one or more pH's, stability atone or more pH's, stability in oxidizing conditions, Ca²⁺ requirements,specific activity, catalytic rate, catalytic efficiency, activity in thepresence of a phytate, thermal or pH stability in the presence of aphytate, ability to effect peak viscosity in a liquefaction test, and/orability to effect final viscosity in a liquefaction test.

As discussed above, the use of phytases may impact the stability orother properties of alpha-amylases by relieving some inhibition onalpha-amylase activity due to the presence of one or more phytates inthe plant material, e.g. milled grain. Without being limited to anyparticular theory, at least partial removal of the phytates appears toimprove one or more properties of the amylase such that yield or resultsare improved.

Reference amylases are discussed above, and in one embodiment of themethod, the reference amylase is SEQ ID NO: 1 or 2.

In various embodiments, the variant is a S242A, S2421D, S242E, S242F,S242G, S242H, S242L, S242M, S242N, S242Q, or S242T variant. In others,the variant further comprises a sequence modification at one or moreamino acid positions corresponding to amino acid positions 97, 179, 180,193, 319, 349, 358, 416, 428, or 443 of the reference amylase. Moreparticularly, the variant comprises one or more of substitution atpositions as follows: a cysteine at 349, a cysteine at 428, a glutamicacid at 97, an arginine at 97, a glutamic acid at 319, an arginine at319, a glutamic acid at 358, an arginine at 358, a glutamic acid at 443,or an arginine at 443 in various embodiments. Substitution of an N193 ora V416 or both, such as a substitution of N193F or V416G, or both arealso useful in certain variants. As with the other modifications, thedeletion of amino acids 179 and 180 can also be used—alone or incombination with any of the foregoing alterations.

In certain embodiments, the parent alpha-amylase is conveniently SEQ IDNO: 1, 2, 15, or 16, while in others, the parent alpha-amylase is SEQ IDNO: 6, 7, 8, 9, 10, 11, or 12.

In one embodiment, the adding step comprises addition of the phytasebefore the amylase. Preferably, when phytase is added first the slurryis pre-incubated after adding the phytase and before adding thealpha-amylase, e.g., for sufficient time to measurably reduce thephytate content.

In various embodiments, the inclusion of the phytase results in anincrease in the thermostability of the alpha-amylase relative to acomparable method that does not include contacting the slurry withphytase.

In other applications, the phytase and the amylase are present in asingle blend, such as a commercial blend, before adding to the slurry.In one presently preferred embodiment, the phytase has the amino acidsequence of SEQ ID NO: 17.

In another of its several aspects, this disclosure provides methods ofproducing a fermentable substrate from a starch-containing slurrycomprising milled grain. The method comprises the steps of:

-   -   a) contacting the starch-containing slurry with at least one        phytase and at least one alpha-amylase in an amount sufficient        to produce a fermentable substrate from the starch; and    -   b) incubating the starch slurry under conditions permissive of        activity of the phytase and the alpha-amylase for a time that        allows production of the fermentable substrate; wherein when the        contact with the phytase is initiated before the amylase, the        slurry is incubated at a temperature that is about 0-30° C. less        than the gelatinization temperature prior to contacting the        slurry with the amylase, after which the temperature is raised        above the gelatinization for a time effective to hydrolyze the        starch.

In various embodiments, the contact with the phytase and thealpha-amylase is initiated at, or about, the same time, or separately inany order. The alpha-amylase used in such methods is a variant amylasecomprising an amino acid sequence at least 95% identical to that of aparent AmyS-like alpha-amylase, and having a substitution at an aminoacid position corresponding to position 242 of a referencealpha-amylase, said variant having detectable alpha-amylase activity.

The reference amylase is SEQ ID NO: 1 or 2, and the variant is a S242A,S242D, S242E, S242F, S242G, S242H, S242L, S242M, S242N, S242Q, or S242Tvariant in certain embodiments.

Also provided herein are kits comprising, in one or more packagesprovided as a unit:

i) at least one variant amylase comprising an amino acid sequence atleast 95% identical to that of a parent AmyS-like alpha-amylase, andhaving a substitution at an amino acid position corresponding toposition 242 of a reference alpha-amylase, said variant havingdetectable alpha-amylase activity, and

ii) at least one additional enzyme.

The kits further comprising instructions for using the enzymes in auseful process involving enzymatic cleavage of starch molecules. Thekits can also further comprise one or more additional enzymes,acidulants or other compounds for adjusting the pH of a starch slurry,nutrients, cofactors, and the like.

This disclosure includes further detail in the following examples, whichare not in any way intended to limit the scope of what is claimed. Thefigures are integral parts of the specification and descriptionprovided. All references cited are herein specifically incorporated byreference for all that is described therein. The following examples arethus offered to illustrate, but not to limit what is claimed.

EXAMPLES Example 1 Construction of Variants

The variants at position S242 of the mature sequence of AmyS wereconstructed using site directed mutagenesis. The template formutagenesis was methylated pHPLT-AmyS (see FIG. 2) using dam-Methylasefrom New England Biolabs (Massachusetts). Degenerate primers(S242F(forward) and S242R(reverse) SEQ ID NOS: 17 and 18 respectively,given below) were synthesized and diluted to 10 μM at Operon(Huntsville, Ala.) with complementary forward and reverse sequences bothcontaining a 5′ phosphate group for ligation in the reaction. Thesequence of the parent alpha-amylase is SEQ ID NO: 2. Libraries werecreated with the Stratagene Quik-Change™ Multi-site kit (Stratagene, LaJolla Calif.) using oligonucleotide primers randomized with NN(G/C) atthe target position. The selected amino acid (i.e., S242) was randomlyreplaced with all 19 possible alternatives.

S242 Primers for Mutagenesis:

S242 F: SEQ ID NO: 17 5′[Phos]GTCAAGCATATTAAGTTCNNSTTTTTTCCTGATTGGTTG 3′S242 R: SEQ ID NO: 18 5′[Phos]CAACCAATCAGGAAAAAASNNGAACTTAATATGCTTGAC 3′The reaction was performed as follows:

QUIK-CHANGE Reaction:

The reaction consisted of 18 μL of sterile distilled H₂O, 2.5 μL of 10×buffer from the kit, 1 μL dNTPs from the kit, 1.25 μL of the forwardprimers (of 10 μM stock), 1.25 μL of the reverse primers (of 10 μMstock), 1 μL of pHPLT-AmyS plasmid DNA as template (˜70 ng), and 1 μL ofthe enzyme blend from the kit for a total of 26.5 μL.

Cycling Conditions:

The cycling conditions were 95° C. for 1 min once, then 95° C. for 1min, 55° C. for 1 min, 65° C. for 10 min for 25 cycles.

One microliter Dpn I (10 U/μL) was added to the Multi-site Quik-Change™reaction mixture and incubated at 37° C. for 18 hours and then another0.5 μL was added for an additional 3 hours.

One microliter of DpnI digested reaction was used as template forrolling circle amplification with the TEMPLIPHI amplification kit(Amersham Biosciences, Piscataway, N.J.) and the reaction was performedaccording to the Amersham protocol. One microliter of rolling circle DNAwas transformed into 100 μL of Bacillus subtilis competent cells (2protease deleted B. subtilis strain (AaprE, AnprE,amyE::xylRPxylAcomK-phleo)) and shaken at 37° C. for 1 hour. The entiretransformation was next plated on LA+10 ppm Neo+1% insoluble starchplates (25 μL one plate, 75 μL on another plate) and incubated overnightat 37° C. Ninety-six transformants were picked into 150 μL of LB+10 ppmNeo in a micro-titer plate and grown overnight at 37° C. The overnightplate was stamped onto a large LA+10 ppm Neo+1% insoluble starch platewith a 96 pin replicating tool and submitted to Quintara Biosciences(Berkeley, Calif.) for colony PCR and sequencing.

After variant sequences were determined, the variants were picked into a96 well micro-titer plates containing 125 μL of LB+10 ppm Neo, arrayingthe variants into a quad format with controls. The arrayed micro-titerplate was grown for 6 hours at 37° C. and 250 rpm. Using a replicatingtool (Enzyscreen, Leiden, The Netherlands) the micro-titer culture platewas used to inoculate a new micro-titer plate (micro-titer plate andplate lids from Enzyscreen, Leiden, The Netherlands) containing 150 ulof MBD medium for protein expression (G. Vogtentanz et al., A Bacillussubtilis fusion protein system to produce soybean Bowman-Birk proteaseinhibitor, Prot. Expr. & Purif. 55 (2007) 40-52) and supplemented with 5mM CaCl₂ for protein expression. Expression plates were grown for 64hours at 37° C., 250 rpm, and 70% humidity. Expression cultures werenext filtered through a micro-filter plate (0.22 μm, Millipore,Billerica, Mass.) and screened for improved thermostability (see Example3).

Example 2 Expression, Purification & Characterization of Variants

Colonies were streaked from the microtiter plates from Example 1 ontostarch plates with 10 ppm Neomycin. The plates were incubated overnightat 37° C. and singles colonies were picked and used to inoculate shakeflasks (250 mL with 25 mL media) containing media (see below) and 20 ppmNeomycin. The cultures were grown up at 37° C., 275 rpm, for about 8 hrs(till an OD (600 nm) of 2.0 was reached). The culture broths were mixedwith 50% glycerol at 2:1 ratio, put into individually-labeled culturevials and frozen at −80° C. Subsequent production of the selectedalpha-amylases were made from these glycerol stocks.

Fermentations for amylases were carried out in 500 mL shake flasks grownat 37° C. for 60 hours in minimal MOPS culture medium (Neidhardt et al.,J. Bacteriol. 119(3): 736-747, 1974) with 1% (w/v) Soytone. Enzymes werepurified from the fermentation broth using hydrophobic interactionchromatography as follows: the broth was concentrated 10-fold thendiluted back to its original volume with 50 mM MES, 2 mM CaCl₂, pH 6.8with 1M ammonium sulfate, then sterile-filtered using glass fiberfilter. Samples were then loaded onto PHENYL SEPHAROSE FF high densitycolumn (20×95 mm; Amersham, GE Healthcare Bio-Sciences, Sweden)pre-equilibrated with the same buffer. Non-amylase proteins were removedwith 10 column volumes of the same buffer without ammonium sulfatefollowed by 5 column volumes of water. Enzymes of interest were elutedwith 50 mM MES, 2 mM CaCl₂, pH 6.8 containing 40% propylene glycol.

Protein concentrations were determined either with a standardquantitative SDS page gel densitometry method or using an activity assayusing a standard amylase assay kit from Megazyme (Wicklow, Ireland). Astandard curve generated using purified amylase (Bacillus 707 amylase;SEQ ID NO: 6) was used for comparing assay data.

Example 3 Determination of Altered Properties: Thermal Stress

This example shows that the variants described herein may have analtered property relative to the parent alpha-amylase. A high-throughputthermal stability screen of G. stearothermophilus alpha-amylase (AmyS)variants was carried out.

After an initial investigation, heat-stress conditions were chosen suchthat the wild-type enzyme showed approximately 40% of its initial(pre-stress) activity after the heat stress (i.e., (activity after heatstress)/(activity before heat stress) was approximately 0.4). Librariesof mutants were screened in quadruplicate, and potential winners wereidentified as those that showed residual activity after heat stress thatwas at least two standard deviations more than the average residualactivity of the wild-type enzyme.

Amylase expression was approximately 100 ppm in the culture supernatantsof the expression plates. After 60-65 hours of growth at 37° C. in ahumidified shaker (250 rpm and 70% relative humidity), the culturesupernatants were clarified to remove cellular material using filterplates. The clarified supernatants were diluted 10-fold into buffercontaining 50 mM NaOAc/2.6 mM CaCl₂/0.002% Tween-20, pH 5.8., to a finalconcentration of approximately 10 ppm. One aliquot of each supernatantwas further diluted to 0.02 ppm, for determination of activity of theenzyme variants as described below using a fluorescently-labeled cornstarch substrate. A second aliquot of each supernatant was subjected toa 30 minute heat stress at 95° C. in a thermocycler then diluted to 0.02ppm in 50 mM NaOAc/2.6 mM CaCl₂/0.002% Tween-20, pH 5.8 and assayed forresidual activity using the fluorescent substrate and assay describedbelow.

Amylase activity was determined using the amylase ENZCHECK ULTRA AMYLASEassay kit essentially as described by the manufacturer (Invitrogen, SanDiego Calif.). Final concentration of the amylase in the assay wasapproximately 0.02 ppm. Assay buffer was 50 mM NaOAc/2.6 mM CaCl₂/0.002%Tween-20, pH 5.8. The substrate was BODIPY fluorescence dye conjugated100 μg/mL DQ™ starch from corn (Invitrogen, Eugene, Oreg.). Increasedfluorescence, indicating amylase activity, was measured using aSpectraMAX M2 (Molecular Devices, Sunnyvale, Calif.). The reaction wasmonitored at room temperature for 5 minutes with the instrumentrecording in kinetic mode. Excitation wavelength was 485 nm; emissionwas monitored at 520 nm with a cutoff filter at 515 nm.

The wild-type AmyS (Xtra) showed 33-43% residual activity after beingsubject to thermal stress for 30 minutes at 95° C. AmyS variants, S242Aand S242Q, retained 55-65% and 70-80% residual activities, respectively,following the same thermal stress conditions. See FIG. 3 and Table 3-1.These residual activity measurements indicate the two variants are morethermostable than the wild-type alpha-amylase.

TABLE 3-1 Percent residual activities of each variant. Wild-type(SPEZYME XTRA). Each plate includes SPEZYME ETHYL and SPEZYME XTRA ascontrols as indicated. Variant % Residual Activity Avg Std. Dev A 65.053.4 48.5 71.1 59.5 10.4 C 35.9 24.5 27.3 29.6 29.3 4.9 D 52.2 32.6 38.543.3 41.6 8.3 E 40.2 53.3 33.2 51.8 44.6 9.6 F 41.7 31.8 30.1 31.7 33.85.3 G 34.3 27.1 27.4 37.5 31.6 5.2 H 22.6 20.5 16.2 17.8 19.3 2.8 I 36.226.9 19.7 25.5 27.0 6.8 K 22.3 22.6 23.3 23.0 22.8 0.5 L 26.1 29.6 30.627.8 28.5 2.0 M 48.8 46.6 40.5 35.9 42.9 5.9 N 32.0 29.0 24.6 35.1 30.24.5 P 7.2 7.7 6.4 5.7 6.7 0.9 Q 61.0 65.7 49.1 69.3 61.3 8.8 R 14.5 14.311.7 11.7 13.0 1.5 wildtype 44.3 27.1 29.2 35.5 34.0 7.7 T 24.6 25.427.7 21.5 24.8 2.5 V 17.5 25.9 22.1 23.9 22.3 3.6 W 5.0 6.3 3.9 7.0 5.61.4 Y 18.5 13.5 14.2 16.5 15.7 2.3 Ethyl 111.8 77.3 84.3 66.7 85.0 19.2Xtra 27.1 36.1 40.7 25.2 32.3 7.4

Example 4 Determination of Altered Properties: DSC

Spezyme Xtra, S242A, S242E, and S242Q were purified from shake flaskfermentation broth (see Example 2) using hydrophobic interactionchromatography. The protein was eluted from the column in purified formusing 50 mM MES, pH 6.8, containing 40% propylene glycol and 2 mM CaCl₂.

Excessive heat capacity curves were measured using an ultrasensitivescanning high-throughput microcalorimeter, VP-CAP DSC (MicroCal, Inc.,Northampton, Mass.). The standard procedure for DSC measurements and thetheory of the technique has been published (Freire, E., “DifferentialScanning Calorimetry,” Methods. Mol. Biol. 41, 191-218, 1995).Approximately 500 μL of 0.5 mg/ml wild-type Bacillus stearothermophilusα-amylase or variant S242A, S242E, and S242Q (both in the absence and inthe presence of 2 mM calcium chloride) were scanned over a 30-120° C.temperature range. The same sample was then re-scanned to check thereversibility of the process. For α-amylase the thermal unfoldingprocess was irreversible. The buffer used was 10 mM sodium acetate, pH5.5. A 200° C./hr scan rate was used to minimize any artifacts that mayhave resulted from aggregation. The thermal midpoint (T_(m)) of the DSCcurves was used as an indicator of the thermal stability of the testedprotein. Table 4-1 shows the thermal melting points for the amylaseproteins tested. The thermal melting curves and the melting points forthe wild-type and variant amylases are shown in FIG. 5.

The thermal unfolding for the amylase variants S242A, S242E, and S242Qin the absence and presence of 2 mM calcium chloride show considerableincrease in the melting points for the variants when compared to thatfor the wild-type. In the absence of added calcium chloride, thewild-type amylase has a thermal melting point of 100.8° C. whilst theT_(m)'s for S242A, S242E, and S242Q are 106.5° C., 107.8° C., and 110.1°C., respectively. Thus, the substitution of S242 with A results in anincrease in the T_(m) of 5.7° C., the substitution of S242 with Eresults in an increase in the T_(m) of 7.0° C., and the substitution ofS242 with Q results in an increase in the T_(m) of 9.3° C.

In the presence of 2 mM calcium chloride, the wild-type amylasedisplayed a thermal melting point of 106.8° C. whilst the T_(m)'s forS242A, S242E, and S242Q were 111.8° C., 112.2° C. and 113.8° C.,respectively. Thus, relative to measurements in the absence of calcium,in the presence of 2 mM calcium chloride, all four proteins hadincreased T_(m) values. The increase in T_(m) for wild-type and theS242A variants in the presence of calcium was 6° C. and 5.3° C.,respectively. The increase in T_(m) for the S242E variant was 4.4° C.The increase in T_(m) for the S242Q variant was 3.7° C. This suggeststhat the S242Q variants is stabilized less by calcium, or the variant isless dependent on calcium for stability. The increase in the T_(m) ofthe S242A, S242E, and S242Q relative to wild-type in the presence ofcalcium chloride was 5° C., 5.4° C., and 3° C., respectively. Thissuggests that the thermodynamic properties of the variants differ fromthose of the wild-type, or Spezyme Xtra. This observation was consistentwith its enhanced performance in application studies (see Example 5).

TABLE 4-1 Tm (° C.) for various amylases by DSC Tm (No Ca²⁺) ΔT (° C.)Tm (w/2 mM Ca²⁺) ΔT (° C.) Spezyme 100.8 106.8 Xtra S242A 106.5 5.7111.8 5.7 S242E 107.8 7.0 112.2 5.4 S242Q 110.1 9.3 113.8 7.0

Example 5 Activity Profiles

This example shows that the tested variants have altered activityprofiles relative not only to the parent alpha-amylase but also to anindustry standard enzyme. Protein determinations were made on purifiedor plate samples. The variants and standard alpha-amylases were eachassayed on the basis of equal protein concentration.

Either plate or purified variants were diluted to approximately 20 ppmusing pH 5.6 malic acid buffer. The substrate consisted of 15%cornstarch in the same 50 mM malic acid buffer, pH 5.6. Four hundredmicroliters of the starch suspension was equilibrated to 70° C. for 2.5minutes. Then 7 μL of the diluted enzyme was quickly added to theequilibrated starch at a final protein concentration of about 0.36 ppm.The reaction mix was then put into a pre-heated 85° C. shaking heatingblock and mixed at 300 rpm. The reactions were quenched with 50 μL of125 mM NaOH at predetermined time intervals. The reaction tubes werespun and the supernatant was diluted 10 fold into 10 mM NaOH, foranalysis of DP profile by HPAEC-PAD.

Reactions were set up for 4, 10 and 20 minutes. The 4 min reactionprovides an indication of the enzyme initial conversion of product tosubstrate; the 10 minute reaction provides an indication of the enzyme'sthermal activity, and the 20 minute reaction provides an indication ofthe enzyme's thermal stability.

Total area from DP2 to the end of the HPLC run was integrated, anddivided by the total protein and reaction time. The results are providedin FIGS. 6 and 7.

Example 6 Liquefaction in the Viscometer

This example shows that the S242A and S242Q variants, which showedaltered residual activity relative to the wild-type parent, also havealtered performance relative to the parent alpha-amylase. The variantalpha-amylases of Example 2 were purified and characterized for totalprotein and specific activity before testing in the viscometer.

Viscosity reduction of corn flour due to the action of the alpha-amylasewas monitored using a HAAKE VISCOTESTER 550 instrument. The substrateslurry was made up fresh daily in batch mode with 30% corn flour drysolids. The pH was adjusted to 5.8 using sulfuric acid. Fifty (50) g ofthe slurry (15 g dry solids) was weighed out and pre-incubated, withstirring, for 10 minutes to warm up to 70° C. Upon alpha-amylaseaddition, the temperature was immediately ramped up from 70° C. to 85°C. with a rotation speed of 75 rpm. Once the temperature of the slurryand enzyme mixture reached 85° C., the temperature was held constant.Viscosity was monitored for an additional 30 minutes. The viscosity wasmeasured throughout the run and reported in μNm. Wild-type AmyS, S242A,and S242Q were each assayed on an equal protein basis at two proteinconcentrations (20 and 30 μg/50 g of corn flour slurry).

The viscometer application showed that both AmyS variants, S242A andS242Q, had better performance than the benchmarkalpha-amylases—Liquozyme SC, Ethyl, and Xtra. The variants exhibitedboth a low peak viscosity, characteristic of Xtra, as well as a lowfinal viscosity, characteristic of Liquozyme SC and Ethyl. When loadedat the lower protein concentration (20 μg total protein), the differencebetween the lower peak viscosity of the variants compared to that ofLiquozyme SC was even more evident. See FIGS. 9, 10 and 11.

Example 7 Liquefaction in a Jet Cooker

Whole ground corn was slurried to a 32% (dry solids corn) slurry byusing a 70:30 ratio of water to thin stillage. The slurry pH wasadjusted to pH 5.8 with 10 N NaOH. The slurry was heated to 70° C. (158°F.) using water and steam in a jacketed kettle. The liquefaction enzymes(SPEZYME Xtra, LiquozymeSC, or S242Q) were added and the slurry washeated to 85° C. (185° F.) over approximately 10 minutes. After theslurry reached 85° C., it was incubated 10 additional minutes at thattemperature. The slurry was passed through a jet-cooker maintained at107° C. (225° F.) with a 3 minute hold time using a pilot plant jetcooker (equipped with an M103 hydro-heater from Hydro-Thermal Corp.,Waukesha, Wis.). The liquefact was collected from the jet and placed inan 85° C. water bath. A second dose of liquefaction enzyme was addedpost-jet. The liquefact was continuously stirred and held at 85° C. for90 minutes. Samples were collected at 0, 30, 60 and 90 minutes. Allpost-jet samples were tested for DE (using the Schoorls method), andviscosity (Brookfield-type viscometer (Lab-Line Instruments Inc.,Melrose Park, Ill.), spindle 3 at 20 rpm). Dosing of liquefactionenzymes pre- and post-jet are indicated in the following figures as“X+Y” where X represents the number of units of enzyme added before thejet, and Y represents the number of units added to the liquefact afterit passes through the jet cooker. Results are shown in FIGS. 12 and 13.

Example 8 Effect of Removal of Phytic Acid Inhibition on Alpha-AmylaseThermostability

The effect of the removal of phytic acid inhibition on thethermostability of liquefying thermostable alpha-amylases was studied.

A. No Jet Cooking (Single Enzyme Dose)

A slurry of whole ground corn (obtained from Badger State Ethanol,Monroe, Wis.) was made with water containing 30% v/v thin stillage to afinal concentration of about 32% ds. Corn solids were prepared in ajacketed kettle. The slurry was mixed well and the pH was measured (pH5.2). No pH adjustment was made. The slurry was mixed in a jacketedkettle and brought to the pretreatment temperature of 70° C. Just priorto reaching 70° C., the liquefying enzyme, i.e., an alpha-amylase (4 AAUper gram ds corn), was added. Identical slurries were treated, one withand one without added phytase (4 FTU per gram ds corn), to begin theincubation or primary liquefaction step. The slurry was incubated for 30minutes in the presence of the amylase with or without added phytase.The phytase used in this experiment was BP-17. Although the phytase wasadded at the same time as the alpha-amylase in this example, it may beadded at other times, such as prior to the amylase.

The treated slurry was then placed in a water bath maintained at 90° C.to begin the secondary liquefaction (2° liquefaction) step. Samples ofeach of the treated slurries (amylase with or without phytases) weretaken at 0, 30, 60 and 90 minutes for viscosity (by Brookfield) and DE(by Schoorls) testing. The results are shown in FIGS. 14 and 15.

B. With jet cooking (Split Enzyme Dose)

A slurry of whole ground corn (obtained from Badger State Ethanol,Monroe, Wis.) was made with water containing 30% v/v thin stillage to afinal concentration of about 32% ds. Corn solids were prepared in ajacketed kettle. The slurry was mixed well and the pH of the slurry wasmeasured (pH 5.2). This slurry was mixed in a jacketed kettle andbrought to 70° C. Just prior to reaching 70° C., the liquefying enzyme,i.e., an S242Q alpha-amylase variant (3 AAU per gram ds corn), was addedto begin the incubation, or primary liquefaction step. Identicalslurries were incubated for 30 minutes in the presence of thealpha-amylase, with or without added phytase (4 FTU per gram ds corn).

Although the phytase was added at the same time as the alpha-amylase inthis example, it may be added at other times, such as prior to theamylase.

Each incubated slurry was passed through a jet cooker (225° F.; 107.2°C.) that was preheated to the desired temperature using steam and water.The slurry was sent through the jet at the maximum speed (1.5 setting)of about 4 liters/minute. Use of the hold coil resulted in a hold timeof just over 3 minutes. After all of the water was displaced and thedesired temperature held steady, an aliquot of solubilized corn mash wascollected and placed in a secondary bath (w/overhead stirring) at 85° C.to begin the secondary liquefaction step (2° liquefaction). A seconddose of the S242Q (1 AAU/gm ds) was added and the liquefaction continuedfor an additional 90 minutes. Samples of each slurry (amylase with orwithout phytases) were taken to test for viscosity (by Brookfield) andDE (by Schoorls) at 0, 30, 60, and 90 minutes.

The resultant liquefact was used in Example 10B.

C. Jet Cooking, Conventional

A slurry of whole ground corn (obtained from Badger State Ethanol,Monroe, Wis.) was made with water containing 30% v/v thin stillage to afinal concentration of about 32% ds. Corn solids were prepared in ajacketed kettle. The slurry was mixed well and the pH of the slurry wasmeasured (pH 5.2). The pH was adjusted to pH 5.8 with dilute NaOH. Theslurry was mixed in a jacketed kettle and brought up to the pretreatmenttemperature of 70° C. Just prior to reaching 70° C., the liquefyingenzyme, i.e., an S242Q alpha-amylase variant (3 AAU per gram ds corn),was added to begin the incubation or primary liquefaction step. Theslurry was incubated for 30 minutes in the presence of the alpha-amylasewithout added phytase.

The incubated slurry was passed through a jet cooker (225° F.; 107.2°C.) that was preheated to the desired temperature using steam and water.The slurry was sent through the jet at the maximum speed (1.5 setting)of about 4 liters/minute. The hold coil resulted in a hold time of justover 3 minutes. After all of the water was displaced and the desiredtemperature held steady, an aliquot of solubilized corn mash wascollected and placed in a secondary bath (overhead stirring) at 85° C.to begin the secondary liquefaction step (2° liquefaction). A seconddose of the S242Q alpha-amylase variant (1 AAU/gm ds) was added and theliquefaction continued for an additional 90 minutes. Samples were takenat 0, 30, 60, and 90 minutes to test for viscosity (by Brookfield) andDE (by Schoorls). The above experiment was conducted at a slurry pH of5.5. See FIG. 21.

The resultant liquefact was used in Example 10A.

D. Results with and Without Jet Cooking

Addition of BP-17 phytase during incubation (primary liquefaction)reduced the phytic acid content of the whole ground corn from 0.60% dscorn to 0.09% ds corn (>85% reduction) (FIG. 20). It is also evidentfrom FIGS. 14 and 15 that the alpha-amylases were inactivated at a jetcooking temperature of 225° F. (107° C.) based on DE development orviscosity reduction. However, the inclusion of phytase prior to jetcooking resulted in a significant increase in the thermostability of thealpha-amylases, as shown by DE progression and viscosity reduction at90° C. during the secondary liquefaction step. Similar results were seenwith jet cooking (data not shown) as shown in FIGS. 14 and 15. Withoutbeing limited to any particular theory of operation, it is believed thataddition of the phytase helps to minimize, reduce, or eliminate phyticacid inhibition of the amylase activity.

Example 9 Effect of BP-17 Phytase Concentration on Alpha-AmylaseStability at Low pH

The increase in the thermostability of alpha-amylase due to the removalof the phytic acid inhibition of alpha-amylase was studied. The phyticacid was hydrolyzed using phytase prior to the secondary liquefaction ofwhole ground corn and the improvement in the pH stability at low pH wasdetermined.

In a typical experiment, whole ground corn was slurried to a 32% (dscorn) by using a 70:30 ratio of water and thin stillage. The slurry pHwas pH 5.2. The slurry was heated to 70° C. using water and steam in ajacketed kettle. The liquefaction enzyme, i.e., the S242Q alpha-amylasevariant (4 AAU/gm ds corn), and varied concentrations of BP-17 (0-12FTU/gm ds corn) were added. The slurry was pretreated by holding thetemperature at 70° C. for 45 minutes. The slurry was then placed in a90° C. water bath. The liquefact was continuously stirred and held at90° C. for 90 minutes. Samples were collected at 0, 30, 60 and 90minutes. All samples were tested for DE (using the Schoorls method), andfor viscosity (Brookfield viscometer, spindle 2 at 20 rpm). The DEprogression and viscosity data are summarized in FIGS. 16-17.

The results showed that the addition of phytase resulted in asignificant increase in the pH stability (at low pH) for amylaseactivity, as evidenced by a steady increase in the DE progression at 90°C., with a concomitant decrease in the viscosity of the liquefact (seeFIGS. 16-17). This may be due to reduction of phytic acid inhibition ofthe alpha-amylase. The data show that the S242Q alpha-amylase variantcan be successfully used in the liquefaction process for whole groundcorn at a pH 5.2 in the presence of an added phytase. In FIG. 20, it canbe seen that the rate of DE progression increases with the increasedaddition of phytase, and reaches a maximum at 4 FTU/gm ds. These resultsmay indicate that phytase increases the thermostability of the S242Qalpha-amylase variant by removing phytic acid from the slurry.

Example 10 Effect of pH

The effect of pH on the S242Q alpha-amylase variant was studied in thisexample.

In a typical experiment, whole ground corn was slurried to a 32% (dscorn) by using a 70:30 ratio of water and thin stillage. The slurry pHwas pH 5.2. The pH was lowered to between 4.2 and 4.8 using H₂SO₄. Theslurry was heated to 70° C. using water and steam in a jacketed kettle.The liquefaction enzyme, i.e., the S242Q variant (4 AAU/gm ds), andBP-17(4 FTU/gm ds) were added and the slurry was pretreated by holdingthe temperature at 70° C. for 45 minutes. The slurry was then placed ina 90° C. water bath. The liquefact was continuously stirred and held at90° C. for 90 minutes. Samples were collected at 0, 30, 60 and 90minutes. All samples were tested for DE (using the Schoorls method), andfor viscosity (Brookfield viscometer, spindle 2 at 20 rpm). The DEprogression and viscosity data are summarized in FIGS. 18-19.

The DE decreased with decreasing pH from 5.2 to 4.5. The amylase enzymewas completely inactivated at pH 4.2.

Example 11 Effect on Ethanol Production

Liquefacts were used as fermentation feedstocks in ethanol fermentationfor alcohol production. A slurry of whole ground corn (obtained fromBadger State Ethanol, Monroe, Wis.) was mixed with water containing 30%v/v thin stillage to a final concentration of about 32% ds.

A. Conventional Process

The liquefact from Example 8, Part C was used (Liquefact A).

The pH of the secondary liquefact was adjusted to 4.2 using H₂SO₄ priorto the simultaneous saccharification and fermentation (SSF) stage.

B. Low pH, Jet Cooking (Split Dose)

The liquefact from Example 8B was used (Liquefact B). No pH adjustmentwas done prior to SSF.

C. Simultaneous Saccharification and Fermentation

In each experiment, tare weights of the vessels were obtained prior topreparation of media. A 32% DS corn liquefact (2 liters) was placed in a2 L flask. Red Star Ethanol Red yeast (RED STAR (Lesaffre)) inoculumswere prepared by adding 10 grams of yeast and 1 gram of glucose to 40grams of water under mild agitation for one hour. Five mls of eachinoculum was added to equilibrated fermentors, followed by the additionof G Zyme™ 480 Ethanol (Danisco US Inc, Genencor Division) at 0.4 GAU/gds corn, to initiate the simultaneous saccharification and fermentation.The initial gross weight was noted and the flask was placed in a waterbath maintained at 32° C. Samples were taken at different intervals oftime and analyzed for carbohydrate and ethanol content using HPLC.Fermentations were also carried out using one kilogram of eachliquefact. Weight loss during fermentation was measured at differentintervals of time. The alcohol was determined based on the weight lossdue to loss of carbon dioxide. At the conclusion of the fermentation, afinal gross weight was obtained. The broth was quantitativelytransferred into a 5 L round bottom vessel. Distillation was performedunder vacuum until approximately 800 mls of ethanol were collected in areceptacle containing 200 mls water. The ethanol was diluted to 2 L andwas analyzed by HPLC. The weight and DS of the still bottoms wasobtained prior to drying. Residual starch analysis was performed on theDDGS. Stoichiometric calculations were performed based on weight loss,distillation, and residual starch analysis.

Ethanol Calculation Using CO₂ Weight Loss:EtOH production (mmol)=CO₂ loss (g)/88EtOH production (g)=(CO₂ loss (g)/88)*92=>CO₂ loss (g)*1.045EtOH production (ml)=((CO₂ loss (g)/88)*92)/0.789=>CO₂ loss (g)×1.325

Table 11 summarizes a comparison of sulfate and phytic acid content inDDGS from a conventional process with that from the process with no pHadjustment. The data show a major difference in free sulphate and phyticacid content between the two processes. Addition of phytase with thethermostable alpha-amylase in the incubation resulted in the DDGS withreduced phytic acid content, higher available (free) phosphate andreduced sulfate. Thus, the process with no pH adjustment confers pHstability at low pH for liquefying thermostable alpha-amylases in thestarch liquefaction.

TABLE 11 Alcohol yield DDGS, % ds Gallons/ Phytic Free Liquefactionconditions Bushel Starch acid % IP 6 Phosphate Sulphate* ConventionalProcess-pH 5.8 2.70 7.25 0.6 100 1.20 1.92 (Liquefact A) No pHadjustment-Process, pH 2.69 9.28 0.2 0 1.33 0.23 5.2 3 + 1 AAU (Splitdose), 4 FTU BP-17, with jet cooking, 225° F. (Liquefact B) *mg/g ds

Example 12 Additional Methods

The following assays were used in the Examples. Deviations from theprotocols provided below are generally indicated in the Examples. Inthese experiments, a spectrophotometer was used to measure theabsorbance of the products formed during the reactions.

A. Protein Content Determination

BCA (Bicinchoninic Acid) Assay

BCA (Pierce) assay was used to determine the protein concentration insamples on microtiter plate (MTP) scale. The chemical and reagentsolutions used were: BCA protein assay reagent, and Pierce dilutionbuffer (50 mM MES, pH 6.5, 2 mM CaCl₂, 0.005% TWEEN®-80). The equipmentincluded a SpectraMAX (type 340; Molecular Devices) MTP reader. The MTPswere obtained from Costar (type 9017).

Two-hundred (200) μL BCA Reagent was pipetted into each well, followedby 20 μL diluted protein. After thorough mixing, the MTPs were incubatedfor 30 minutes at 37° C. Air bubbles were removed before the opticaldensity (OD) of the solution in the wells was read at 562 nm. Todetermine the protein concentration, the background reading wassubtracted from the sample readings. The OD₅₆₂ was plotted for proteinstandards (purified enzyme) to produce a standard curve. The proteinconcentration of the samples were interpolated from the standard curve.

Bradford Assay

The Bradford dye reagent (Quick Start) assay was used to determine theprotein concentration in samples on MTP scale. The chemical and reagentsolutions used were: Quick Start Bradford Dye Reagent (BIO-RAD CatalogNo. 500-0205), Dilution buffer (10 mM NaCl, 0.1 mM CaCl₂, 0.005%TWEEN®-80. The equipment used was a Biomek FX Robot (Beckman) and aSpectraMAX (type 340) MTP reader. The MTPs were from Costar (type 9017).

Two-hundred (200) μL Bradford dye reagent was pipetted into each well,followed by 15 μL dilution buffer. Ten (10) μL of filtered culture brothwere added to the wells. After thorough mixing, the MTPs were incubatedfor at least 10 minutes at room temperature. Air bubbles were blown awayand the OD of each well was read at 595 nm. To determine the proteinconcentration, the background reading (i.e., from un-inoculated wells)was subtracted form the sample readings. The OD₅₉₅ values obtainedprovide a relative measure of the protein content in the samples.

B. Microswatch Assay for Testing Enzyme Performance

The detergents used in this assay did not contain enzymes or the enzymespresent in commercial detergents had been destroyed through heatdeactivation as described elsewhere in this document. The equipment usedincluded an Eppendorf Thermomixer and a SpectraMAX (type 340) MTPreader. The MTPs were obtained from Costar (type 9017).

Detergent Preparation (AATCC HDL; US Conditions)

Milli-Q water was adjusted to 6 gpg water hardness (Ca/Mg=3/1), and 1.5g/l AATCC 2003 standard reference liquid detergent without brightenerwas added. The detergent solution was vigorously stirred for at least 15minutes. Then, 5 mM HEPES (free acid) was added and the pH adjusted to8.0.

Rice Starch Microswatch Assay for Testing Amylase Performance

Test detergents were prepared as described elsewhere in this document.The equipment used included a New Brunswick Innova 4230 shaker/incubatorand a SpectraMAX (type 340) MTP reader. The MTPs were obtained fromCorning (type 3641). Aged rice starch with orange pigment swatches(CS-28) were obtained from Center for Test Materials (Vlaardingen,Netherlands). Before cutting 0.25-inch circular microswatches, thefabric was washed with water. Two microswatches were placed in each wellof a 96-well microtiter plate. The test detergent was equilibrated at20° C. (North America) or 40° C. (Western Europe). 190 μL of detergentsolution were added to each well of the MTP, containing microswatches.To this mixture, 10 μL of the diluted enzyme solution was added. The MTPwas sealed with adhesive foil and placed in the incubator for 1 hourwith agitation at 750 rpm at the desired test temperature (typically 20°C. or 40° C.). Following incubation, 150 μL of the solution from eachwell were transferred into a fresh MTP and read at 488 nm using aSpectraMAX MTP reader to quantify cleaning. Blank controls, as well ascontrols containing microswatches and detergent, but no enzyme, werealso included.

Calculation of Enzyme Performance

The obtained absorbance value was corrected for the blank value (i.e.,obtained after incubation of microswatches in the absence of enzyme).The resulting absorbance was a measure of the hydrolytic activity.

C. Amylase Concentration Determination By Antibody Titration

Alpha-amylase concentration and specific activity was determined, insome cases, by titration with an inhibitory polyclonal antibody.Polyclonal antibodies raised to Bacillus stearothermophilusalpha-amylase (AmyS) were found to be strongly inhibitory of AmyS andthe alpha-amylase from Bacillus sp. TS23 (e.g., the binding is tightenough to produce a linear titration of activity loss). Therefore, thisantibody can be used to measure enzyme concentration, which, in turn, isused to calculate specific activity.

Briefly, the amount of enzyme inhibition produced by several knownconcentrations of antibody is measured. From this information, theconcentration of antibody required for complete inhibition isextrapolated, which is equivalent to the enzyme concentration in thesample. Alpha-amylase activity and inhibition was measured using thefluorogenic BODIPY-starch assay. The buffer was 50 mM MOPS, pH 7.0,containing 0.005% Tween-80.

A polyclonal antibody directed against purified AmyS was raised in arabbit and purified by standard methods. An empirical “apparentconcentration” value of an antibody stock solution was determined bymeasuring the inhibition of a sample of AmyS of known specific activity.The antibody sample was used to determine the concentration and specificactivity of AmyS and TS23t variants. These values were used to createnormalized 96-well enzyme stock plates, in which all of the variantswere diluted to a common concentration.

D. Native Protein Gel Electrophoresis

Electrophoretic mobility of variant protein samples was measured usingthe PHASTGEL system (GE Healthcare) on pre-cast native polyacrylamidegels (PHASTGEL Homogeneous) at either 7.5% or 12.5% concentration.Buffer strips (PHASTGEL Native) were used and consisted of pH 8.8 in0.88 M L-Alanine, 0.25 M Tris buffer. Typical run conditions consistedof 400V for 12.75 minutes with an anode-to-cathode distance of 3.7 cm.

Alternatively, electrophoretic mobility of variant protein samples wasmeasured on 1 mm-thick 0.5-1.5% agarose gels at various pH values (i.e.5.8, 8.0 and 10.0) through a choice of a suitable buffer system. Theelectrophoresis was carried out under non-denaturing conditions. TheCathode-Anode length was 13.9 cm. A sample of 1-2 μg protein was mixedwith 5% glycerol+0.05% bromophenol blue and loaded on each lane. Gelswere run typically for 1 hour at 100V.

Gels were stained with Louisville blue dye dissolved in 10% acetic acidand destained with 10% methanol and 10% acidic acid-in-water. Between 12and 20 protein variants were loaded simultaneously, depending on nativegel system used. As a consequence, the electrophoretic mobility of aprotein variant can be immediately assessed, relative to charge ladderstandards loaded on the same gel.

E. Detergent Heat Inactivation

Heat inactivation of commercial detergent formulas serves to destroy theenzymatic activity of any protein components while retaining theproperties of non-enzymatic components. Thus, this method was suitablefor preparing commercially-purchased detergents for use in testing theenzyme variants. For North American (NA) and Western European (WE) heavyduty liquid laundry (HDL) detergents, heat inactivation was performed byplacing pre-weighed liquid detergent (in a glass bottle) in a water bathat 95° C. for 2 hours. The incubation time for heat inactivation ofNorth American (NA) and Japanese (JPN) heavy duty granular laundry (HDG)detergent was 8 hours and that for Western European (WE) HDG detergentwas 5 hours. The incubation time for heat inactivation of NA and WE autodishwashing (ADW) detergents was 8 hours. The detergents were purchasedfrom local supermarket stores. Both un-heated and heated detergents wereassayed within 5 minutes of dissolving the detergent to accuratelydetermine percentage deactivated. Enzyme activity was tested by thesuc-AAPF-pNA assay.

For testing of enzyme activity in heat-inactivated detergents, workingsolutions of detergents were made from the heat inactivated stocks.Appropriate amounts of water hardness (6 gpg or 12 gpg) and buffer wereadded to the detergent solutions to match the desired conditions (Table12-1). The solutions were mixed by vortexing or inverting the bottles.

TABLE 12-1 Laundry and Dish Washing Conditions Region Form DoseDetergent* Buffer Gpg pH T (° C.) Laundry (heavy duty liquid andgranular) NA HDL 0.78 g/l  P&G TIDE ® 2X 5 mM HEPES 6 8.0 20 WE HDL 5.0g/L Henkel Persil 5 mM HEPES 12 8.2 40 WE HDG 8.0 g/L P&G Ariel 2 mMNa₂CO₃ 12 10.5 40 JPN HDG 0.7 g/L P&G TIDE ® 2 mM Na₂CO₃ 6 10.0 20 NAHDG 1.0 g/L P&G TIDE ® 2 mM Na₂CO₃ 6 10.0 20 Automatic Dish Washing WEADW 3.0 g/L RB Calgonit 2 mM Na₂CO₃ 21 10.0 40 NA ADW 3.0 g/L P&GCascade 2 mM Na₂CO₃ 9 10.0 40 *Abbreviations: Procter & Gamble (P&G);and Reckitt Benckiser (RB).

F. TERG-O-TOMETER Assay for Cleaning Performance Determination

A standard protocol for assessing protein and carbohydrate soil cleaningwas used whereby the soil level on a fabric swatch was measured beforeand after cleaning under standard conditions. The fabric swatchesconsisted of woven cotton fabric soiled with either maize starch, ricestarch or a blood, milk, and carbon black mixture. Swatches werepurchased from Testfabrics, Inc. (West Pittston, Pa.). Maize Starch(EMPA 161) and Blood, Milk, Carbon Black (EMPA 116) technical soils wereproduced by EMPA Test materials AG (St. Gallen, Switzerland). RiceStarch (CFT CS-28) soils were produced by the Center for TestmaterialsBV (Vlaardingen, Netherlands). Each stain was measured before and aftertreatment by optical reflectance using a Minolta Reflectometer CR-410set to a D65 (6500° K.) standard illuminant. The difference in the L, a,b values was converted to total color difference (dE), as defined by theCIE-LAB color space. Cleaning of the stains are expressed as percentstain removal index (% SRI) by taking a ratio between the colordifference before and after washing and comparing it to the differenceof unwashed soils (before wash) to unsoiled fabric.

Cleaning experiments were conducted in a TERG-O-TOMETER (United StatesTesting Co., Hoboken, N.J.) equipped with 6 stainless steel 2 L potsfitted with overhead agitators. Each treatment was conducted in 1 Ltotal volume consisting of either 6 grains per gallon 3:1(calcium:magnesium) water hardness or 12 grains per gallon waterhardness. Detergents used in the wash experiments were 1.5 g/L AATCC HDLWOB 2003 liquid detergent with 5 mM HEPES buffer at pH 8, 0.7 g/L AATCCHDD WOB 1993 granular detergent, 8 g/L IEC A* 60456 granular detergentwith perborate and TAED bleach, or 5 g/L Persil Power Gel liquiddetergent. Enzyme was added directly into the wash solution andreactions were then initiated by addition of either 40 g/L or 200 g/L ofsoiled and ballast fabric. The washing reactions were agitated at 100rpm for 10, 15, or 40 minutes at 20° C., 25° C., 30° C., 40° C., or 50°C. Following cleaning, swatches were rinsed for 3 minutes in tap water,spun in a front-loading washing machine at 1000 rpm to remove excesswater, and dried in a dryer at low heat on a permanent press cycle forapproximately 45 minutes. Comparison of the extent of soil removal wasassessed by reflectometry and expressed as the % soil removal index (%SRI). The control condition did not contain enzyme and the positivecontrol consisted of various doses of benchmark commercial enzymes.

G. BODIPY-Starch Assay for Determination of Amylase Activity

The BODIPY-starch assay was performed using the EnzChek® Ultra AmylaseAssay Kit (E33651, Invitrogen). A 1 mg/mL stock solution of the DQstarch substrate was prepared by dissolving the contents of the vialcontaining the lyophilized substrate in 100 μL of 50 mM sodium acetatebuffer at pH 4.0. The vial was vortexed for about 20 seconds and left atroom temperature, in the dark, with occasional mixing until dissolved.900 μL of assay buffer (50 mM sodium acetate with 2.6 mM CaCl₂ pH 5.8)was added and the vial was mixed by vortex for about 20 seconds. Thesubstrate solution was stored at room temperature, in the dark, untilready to use or at 4° C. For the assay, a 100 μg/mL of working solutionof the DQ substrate was prepared from the 1 mg/mL substrate solution inthe assay buffer. 190 μL of 100 μg/mL substrate solution was added toeach well in a flat-bottom 96-well microtiter plate. 10 μL of eachenzyme sample was added to a well, mixed for 30 seconds using athermomixer at 800 rpm. A blank sample containing buffer and substrateonly (no-enzyme blank) was included in the assay. The rate of change offluorescence intensity was measured (excitation: 485 n, emission: 520μm) in a fluorescence microtiter plate reader at 25° C. for 5 minutes.

H. Corn Flour Hydrolysis for Determination of Amylase Activity

Starch Hydrolysis of Corn Flour Substrate Assay for Enzymatic Activity.Organic corn flour (Azure Farms, lot no. 03227) was evenly spread intoGreiner 96-well microplate, polypropylene, black, flat bottom chimneywells, (Cat. No. 655209), using a solids dispensing device (V&PScientific). 85 μL of 20 mM sodium acetate pH 5.6 were added to eachwell and mixed. A foil seal was applied to the top of the plate and theplate pre-incubated at 70° C. in the Thermomixer for 20-30 minutes.Enzyme samples were diluted in Agilent polypropylene plate (5042-1385)in 20 mM sodium acetate buffer. 11 μL of diluted enzyme samples wereadded to the substrate plate and the plate sealed firmly with anotherfoil. Plates were then transferred to Labnet VorTemp 56 Incubator/Shakerwith metal blocks (Cat. No. S2056A), pre-heated to 95° C. and the shakespeed set to 500 rpm. The incubation was continued for 30 minutes. Atthe end of the incubation, the plates were rapidly cooled in an icebucket and the starch hydrolysis reaction was stopped by addition of 100μL of 0.1N H2SO4 to each well. The plate was mixed briefly and thestarch hydrolysis reaction products were either analyzed by the PAHBAHassay or HPLC.

Colorimetric detection of Soluble Sugar Concentrations from EnzymaticHydrolysis of Corn Flour Substrate. Aliquots of 80 μL of 0.5 N NaOH wereadded to all wells of an empty PCR plate followed by 20 μL of PAHBAHreagent (5% w/v p-hydroxybenzoic acid hydrazide (PAHBAH, Sigma # H9882,dissolved in 0.5 N HCl) and mixed (PAHBAH reaction plate). 10 μL of thestarch hydrolysis reaction supernatants were added to the PAHBAHreaction plate. All plates were sealed and placed in the thermocycler(MJ Research Tetrad), programmed for 2 minutes at 95° C., and thencooled to 20° C. Samples of 80 μL of the developed PAHBAH reactionmixtures were transferred to a read plate and absorbance was measured at405 nm in a spectrophotometer.

HPLC Determination of soluble Sugar Concentrations from EnzymaticHydrolysis of Corn Flour Substrate. Soluble sugar standards (DP1-DP7)obtained from Sigma (St. Louis, Mo.) were all diluted in Milli-Q waterto 100 mg/mL and used for converting peak area for the sugars to actualsugar concentrations. The quenched plate from the starch hydrolysisassay was spun in a Beckman Coulter Allegra 6R Centrifuge for 5 minutesat 3000 rpm 25° C. The supernatant was pipetted from the spun plate andtransferred to a Multiscreen-HV filter plate (Catalog No. MAHVN4550).The filter plate was spun over an Agilent HPLC plate in the HettichRotanta centrifuge for 10 minutes at 6000 rpm 25° C. 50 μL of 0.01Nsulfuric acid mobile phase (0.1N sulfuric acid diluted 10× with Milli-Qwater) was transferred to each well of another clean Agilent HPLC plate.The filtered plate was briefly mixed and 50 μL of the filtrate wastransferred the corresponding wells in the plate with 50 μL per well ofmobile phase. Diluted sugar standards were added to empty wells in theplate to be included in the calibration. The contents were mixed brieflyon a platform shaker and the plate covered with a Nalgene Pre-slit WellCap. The HPLC column (Bio-Rad Aminex HPX-87H column Cat No. 125-0140)was prepared ahead of time with 2L of mobile phase running at a constantflow rate of 0.6 mL/minute. All samples in the plate were run with 20 μLinjection volume and analyzed using AMINEXH.M and RID (refractive index)as the detector. After the run was completed, the flow rate in the HPLCwas dropped down to 0.05 mL/min.

I. Determination of Starch Viscosity Reduction By Alpha-Amylase

In this assay, viscosity reduction of cornstarch substrate solution wasmeasured in a viscometer. The cornstarch substrate slurry was made upfresh in batch mode with 30% corn flour dry solids in distilled waterand adjusted to pH 5.8 using sulfuric acid. For each run, 50 grams ofthe slurry (15 grams dry solids) was weighed out and pre-incubated for10 minutes to warm up to 70° C. Upon amylase addition, the temperaturewas immediately ramped up from 70° C. to 85° C. with a rotation speed of75 rpm. Once the temperature of the slurry and amylase mixture reached85° C., the temperature was held constant and viscosity was monitoredfor an additional 30 minutes.

J. Measurement of Enzyme Binding to Macromolecular Substrates

Binding assays were done to determine substrate binding of Amylase(AmyS) charge ladder variants (charge change=−12 to +12 relative towild-type AmyS) to corn stover and bagasse. Substrates used includedbagasse (sugarcane bagasse from Brazil, dilute-acid pre-treated byNational Renewable Energy Laboratory, washed and buffered at pH 5), AFEX(ammonia fiber expansion corn stover), and PCS (dilute sulfuric acidpre-treated corn stover, washed and adjusted to pH 5). All substrateswere brought to the desired percentage solids prior to use.

Amylase Binding: Amylase charge ladder variants were purified anddiluted to 200 ppm for testing. A 1% cellulose bagasse solution wasprepared in borate buffer (40 mM, pH 8.5, 0.016% Tween-80). 150 μL ofthe bagasse solution was added into each well in a microtiter filtrationplate. 150 μL of borate buffer was added into a set of separate wells,which served as controls. 10 μL of amylase charge ladder variants wasadded into the filtration plate, each condition was in duplicates. Theplate was incubated at room temperature for 2 hours. The filtrate wascollected and amylase activity in the supernatant was measured byBODIPY-starch assay.

Measurement of Enzyme Binding to Microswatches: Amylase variants wereincubated with or without CS-28 rice starch microswatches under standardwash conditions for 30 min. The amount of free enzyme was measured bythe BODIPY-starch assay. The fraction of enzyme bound to themicroswatches was calculated as follows: Fraction bound=(Activity ofenzyme in absence of swatch−Activity of enzyme in presence ofswatch)/(Activity of enzyme in absence of swatch).

Example 13 Amylase Production in B. subtilis

In this Example, production of a mutant truncated form Bacillusstearothermophilus amylase alpha-amylase (having a S242Q mutation and a29 amino acid deletion from the C-terminus; also referred to herein asS242Q) and variants thereof in B. subtilis are described. Transformationwas performed as known in the art (see e.g., WO 02/14490). Briefly, thegene encoding the parent amylases was cloned into the pHPLT expressionvector, which contains the LAT promoter (PLAT), a sequence encoding theLAT signal peptide (preLAT), followed by PstI and HpaI restriction sitesfor cloning.

The coding region for the LAT signal peptide is shown below:

(SEQ ID NO: 19) atgaaacaac aaaaacggct ttacgcccga ttgctgacgc tgttatttgcgctcatcttc ttgctgcctc attctgcagc ttcagca.

The amino acid sequence of the LAT signal peptide is shown below:

MKQQKRLYAR LLTLLFALIF LLPHSAASA (SEQ ID NO: 20)

The amino acid sequence of the mature truncated S242Q amylase with thesubstituted amino acid shown in italics was used as the basis for makingthe variant libraries described herein:

(SEQ ID NO: 21) AAPFNGTMMQ YFEWYLPDDG TLWTKVANEA NNLSSLGITA LWLPPAYKGTSRSDVGYGVY DLYDLGEFNQ KGTVRTKYGT KAQYLQAIQA AHAAGMQVYA DVVFDHKGGADGTEWVDAVE VNPSDRNQEI SGTYQIQAWT KFDFPGRGNT YSSFKWRWYH FDGVDWDESRKLSRIYKFRG IGKAWDWEVD TENGNYDYLM YADLDMDHPE VVTELKNWGK WYVNTTNIDGFRLDAVKHIK FQFFPDWLSY VRSQTGKPLF TVGEYWSYDI NKLHNYITKT NGTMSLFDAPLHNKFYTASK SGGAFDMRTL MTNTLMKDQP TLAVTFVDNH DTEPGQALQS WVDPWFKPLAYAFILTRQEG YPCVFYGDYY GIPQYNIPSL KSKIDPLLIA RRDYAYGTQH DYLDHSDIIGWTREGVTEKP GSGLAALITD GPGGSKWMYV GKQHAGKVFY DLTGNRSDTV TINSDGWGEFKVNGGSVSVW VPRKTT.

The PCR products were purified using QIAQUIK columns from Qiagen, andresuspended in 50 μL of deionized water. 50 μL of the purified DNA wasdigested with HpaI (Roche) and PstI (Roche), and the resultant DNAresuspended in 30 μL of deionized water. 10-20 ng/μL of the DNA wascloned into plasmid pHPLT using PstI and HpaI cloning sites. Theligation mixtures were directly transformed into competent B. subtiliscells (genotype: Δvpr, ΔwprA, Δmpr-ybfJ, ΔnprB). The B. subtilis cellshave a competency gene (comK) which is placed under a xylose induciblepromoter, so xylose was used to induce competency for DNA binding anduptake (see Hahn et al., Mol. Microbiol., 21: 763-775, 1996).

The elements of plasmid pHPLT-AmyS include: pUB110=DNA fragment fromplasmid pUB110 (McKenzie et al., Plasmid 15: 93-103, 1986). Plasmidfeatures include: ori-pUB110=origin of replication from pUB110;neo=neomycin resistance gene from pUB110; Plat=transcriptional promoterfrom B. licheniformis amylase; Pre LAT=signal peptide from B.licheniformis amylase; SAMY 425ss=The coding region for truncated AmyEgene sequence (replaced by the coding regions for each truncated AmyEvariant expressed in this study); and Terminator=transcriptionalterminator from B. licheniformis amylase.

Example 14 Expression of Enzyme Variants

This Example describes the methods used to express various recombinantenzymes of the transformed B. subtilis of the preceding Examples.

Amylase Expression—2 mL Scale

B. subtilis clones containing S242Q (or a variant thereof) expressionvectors were replicated with a steel 96-well replicator from glycerolstocks into 96-well culture plates (BD, 353075) containing 150 μL of LBmedia+10 μg/ml neomycin, grown overnight at 37° C., 220 rpm in ahumidified enclosure. A 100 μL aliquot from the overnight culture wasused to inoculate 2000 μL defined media+10 μg/ml neomycin in 5 mLplastic culture tubes. The cultivation media was an enrichedsemi-defined media based on MOPS buffer, with urea as major nitrogensource, glucose as the main carbon source, and supplemented with 1%SOYTONE and 5 mM calcium for robust cell growth. Culture tubes wereincubated at 37° C., 250 rpm, for 72 hours. Following this incubation,the culture broths were centrifuged for 10 minutes at 3000×g. Thesupernatant solution was decanted into 15 mL polypropylene conical tubesand 80 μL of each sample were aliquoted into 96 well plates for proteinquantitation.

Example 15 Production of Enzyme Variants

This Example describes the production of enzyme charge ladders andcombinatorial charge libraries.

Enzyme Charge Ladders

Multiple protein variants spanning a range of physical properties ofinterest are selected from existing libraries or are generated bysite-directed mutagenesis techniques as known in the art (See e.g., U.S.patent application Ser. Nos. 10/576,331, 11/581,102, and 11/583,334,assigned to Genencor International). This defined set of probe proteinsis then assayed in a test of interest.

Exemplary amylase charge ladder variants are shown in the followingtables and assayed as described herein. In these tables, the chargechange is relative to the parent enzyme.

TABLE 15-1 AmyS-S242Q Charge Ladder AmyS-S242Q Variant Δ ChargeQ97E-Q319E-Q358E-Q443E −4 Q97E-Q319E-Q358E −3 Q97E-Q319E −2 Q97E −1Q97R-Q319E 0 Parent AmyS-S242Q 0 Q97R +1 Q97R-Q319R +2 Q97R-Q319R-Q358R+3 Q97R-Q319R-Q358R +4

Enzyme Combinatorial Charge Libraries (CCL)

Generation of B. stearothermophilus AmyS-S242Q CCL

The AmyS-S242Q plasmid DNA was isolated from a transformed B. subtilisstrain (genotype: AaprE, AnprE, amyE::xylRPxylAcomK-phleo) and sent toDNA2.0 Inc. as the template for CCL construction. A request was made toDNA2.0 Inc. (Mountain View, Calif.) for the generation of positionallibraries at each of the four sites in AmyS-S242Q (S242Q) amylase thatare shown in Table 15-2. Variants were supplied as glycerol stocks in96-well plates.

The AmyS S242Q combinatorial charge library was designed by identifyingthe following four residues: Gln-97, Gln 319, Gln 358, and Gln 443. Afour site, 81-member CCL was created by making all combinations of threepossibilities at each site: wild-type, arginine, or aspartic acid.

TABLE 15-2 S242Q CCL Variants Variant # Q97 Q319 Q358 Q443 Δ Charge  1Q97E Q319E Q358E Q443E −4  2 Q97E Q319E Q358E Q443R −2  3 Q97E Q319EQ358E — −3  4 Q97E Q319E Q358R Q443E −2  5 Q97E Q319E Q358R Q443R 0  6Q97E Q319E Q358R — −1  7 Q97E Q319E — Q443E −3  8 Q97E Q319E — Q443R −1 9 Q97E Q319E — — −2 10 Q97E Q319R Q358E Q443E −2 11 Q97E Q319R Q358EQ443R 0 12 Q97E Q319R Q358E — −1 13 Q97E Q319R Q358R Q443E 0 14 Q97EQ319R Q358R Q443R +2 15 Q97E Q319R Q358R — +1 16 Q97E Q319R — Q443E −117 Q97E Q319R — Q443R +1 18 Q97E Q319R — — 0 19 Q97E — Q358E Q443E −3 20Q97E — Q358E Q443R −1 21 Q97E — Q358E — −2 22 Q97E — Q358R Q443E −1 23Q97E — Q358R Q443R +1 24 Q97E — Q358R — 0 25 Q97E — — Q443E −2 26 Q97E —— Q443R 0 27 Q97E — — — −1 28 Q97R Q319E Q358E Q443E −2 29 Q97R Q319EQ358E Q443R 0 30 Q97R Q319E Q358E — −1 31 Q97R Q319E Q358R Q443E 0 32Q97R Q319E Q358R Q443R +2 33 Q97R Q319E Q358R — +1 34 Q97R Q319E — Q443E−1 35 Q97R Q319E — Q443R +1 36 Q97R Q319E — — 0 37 Q97R Q319R Q358EQ443E 0 38 Q97R Q319R Q358E Q443R +2 39 Q97R Q319R Q358E — +1 40 Q97RQ319R Q358R Q443E +2 41 Q97R Q319R Q358R Q443R +4 42 Q97R Q319R Q358R —+3 43 Q97R Q319R — Q443E +1 44 Q97R Q319R — Q443R +3 45 Q97R Q319R — —+2 46 Q97R — Q358E Q443E −1 47 Q97R — Q358E Q443R +1 48 Q97R — Q358E — 049 Q97R — Q358R Q443E +1 50 Q97R — Q358R Q443R +3 51 Q97R — Q358R — +252 Q97R — — Q443E 0 53 Q97R — — Q443R +2 54 Q97R — — — +1 55 — Q319EQ358E Q443E −3 56 — Q319E Q358E Q443R −1 57 — Q319E Q358E — −2 58 —Q319E Q358R Q443E −1 59 — Q319E Q358R Q443R +1 60 — Q319E Q358R — 0 61 —Q319E — Q443E −2 62 — Q319E — Q443R 0 63 — Q319E — — −1 64 — Q319R Q358EQ443E −1 65 — Q319R Q358E Q443R +1 66 — Q319R Q358E — 0 67 — Q319R Q358RQ443E +1 68 — Q319R Q358R Q443R +3 69 — Q319R Q358R — +2 70 — Q319R —Q443E 0 71 — Q319R — Q443R +2 72 — Q319R — — +1 73 — — Q358E Q443E −2 74— — Q358E Q443R 0 75 — — Q358E — −1 76 — — Q358R Q443E 0 77 — — Q358RQ443R +2 78 — — Q358R — +1 79 — — — Q443E −1 80 — — — Q443R +1 81(parent) Q97 Q319 Q358 Q443 0

Example 16 Enzyme Wash Performance

This Example describes the testing of S242Q variant in a microswatchassay 1.0 μg/ml in AATCC HDL detergent or 5 mM HEPES buffer undervarying ionic strength. The methods provided in Example 12 were used(See, “Rice Starch Microswatch Assay for testing Amylase Performance”and “Corn Four Hydrolysis”).

There is an optimal net charge change for cleaning performance forenzyme in AATCC HDL detergent. Performance is measured in terms ofrelative cleaning performance observed in a rice starch microswatchactivity assay. A value of around 1.0 indicates top cleaning performancein this assay. This is an example of optimizing a protein physicalproperty (e.g., net charge) for improving a given outcome or benefit(e.g., cleaning performance in a liquid laundry detergent). The chargeoptimum identified with this limited set of probe proteins coincideswith the optimum charge observed when measuring the entire chargecombinatorial library. The use of probe proteins is therefore predictiveof the behavior of the entire library.

According to the Debye-Hückel theory (Israelachivili, Intermolecular andSurface Forces, 2^(nd) Edition: With Applications to Colloidal andBiological Systems, Academic Press 2^(nd) Ed. [1992]), electrostaticinteractions are governed primarily by the strength of double-layerforces between interacting species at constant potential or constantcharge (enzymes, substrates, fabric, and detergent), their size, and thedielectric constant of the surrounding medium. In order to characterizethe electrostatic behavior of particles in a complex medium, such as adetergent formulation, their interaction in a reduced environmentpossessing the same Debye screening length is sufficient. This wasaccomplished by choosing a buffer of matching pH and conductivity tothat of the detergent under wash conditions. An appropriate buffer forsuch testing is 5 mM HEPES buffer at pH 8.0 with varying amounts ofindifferent electrolyte, such as NaCl. Addition of 2.5 mM NaCl to thisbuffer matches the pH and conductivity of typical North American washconditions. Addition of a higher concentration of NaCl is representativeof Japanese and European wash conditions, typically higher in ionicstrength due to both increased water hardness and detergentconcentrations.

FIG. 22 shows that positive charge S242Q charge variants are superiorfor cleaning of rice starch microswatch under North American laundryconditions. Likewise negative charge TS23t variants are superior forcleaning of rice starch microswatches in Western European laundryconditions (FIG. 23).

FIG. 24 demonstrates that positive S242Q variants exhibit higherspecific activity for granular corn starch substrates hydrolysis.

Example 17 Thermostability

This Example describes determining the relationship between proteincharge and thermal stability. Alpha-amylase assays were based on BODIPYstarch hydrolysis before and after heating the culture supernatant. Thesame chemical and reagent solutions were used as described in Example12.

Thermal Stability Assay for Alpha-Amylases

The filtered culture supernatants were serially diluted in 50 mM sodiumacetate+2 mM CaCl₂ pH 5.8 with 002% Tween. 10 μL of each diluted culturesupernatant was assayed to determine the initial amylase activity by theBODIPY starch assay. 50 μL of each diluted culture supernatant wasplaced in a VWR low profile PCR 96 well plate. 30 μL of mineral oil wasadded to each well as a sealant. The plate was incubated in a BioRad DNAengine Peltier Thermal Cycler at 95° C. for 30 or 60 minutes dependingon the stability of the parent enzyme. Following incubation, the platewas cooled to 4° C. for 5 min and then kept at room temperature. 10 μLof each sample was added to a fresh plate and assayed to determine thefinal amylase activity by the BODIPY starch assay as described inExample 1.

Calculation of Thermostability

The residual activity of a sample was expressed as the ratio of thefinal absorbance and the initial absorbance, both corrected for blanks.A higher index indicates a more thermally stable variant. This is anexample of optimizing a protein physical property, in this case netcharge, for improving enzyme thermal stability for a liquid laundryapplication.

Thermostability Assay

Thermostability of the variants was assessed as described above.Thermostability winners are listed in Table 17-1. Winners were definedas those having a ratio of mutant residual activity to parent (i.e.,S242Q) residual activity greater than 1.

TABLE 17-1 S242Q CCL - thermal stability winners Mut residual act./WTresidual Variant # 97 319 358 443 act. 2 Q97E Q319E Q358E Q443R 1.12 10Q97E Q319R Q358E Q443E 1.12 13 Q97E Q319R Q358R Q443E 1.36 14 Q97E Q319RQ358R Q443R 1.16 15 Q97E Q319R Q358R 1.37 17 Q97E Q319R Q443R 1.29 18Q97E Q319R 1.11 27 Q97E 1.16 32 Q97R Q319E Q358R Q443R 1.18 37 Q97RQ319R Q358E Q443E 1.29 38 Q97R Q319R Q358E Q443R 1.22 39 Q97R Q319RQ358E 1.21 40 Q97R Q319R Q358R Q443E 1.20 41 Q97R Q319R Q358R Q443R 1.2642 Q97R Q319R Q358R 1.48 43 Q97R Q319R Q443E 1.21 44 Q97R Q319R Q443R1.21 45 Q97R Q319R 1.14 50 Q97R Q358R Q443R 1.14 62 Q319E Q443R 1.26 63Q319E 1.18 64 Q319R Q358E Q443E 1.19 65 Q319R Q358E Q443R 1.28 68 Q319RQ358R Q443R 1.14 70 Q319R Q443E 1.22 73 Q358E Q443E 1.15 74 Q358E Q443R1.15 75 Q358E 1.18

Example 18 Enzyme Performance

This Example demonstrates that enzyme performance may be affected bycharge.

Enzyme performance was assessed using heat inactivated detergents asdescribed above in Example 12. Winners were defined as those havingPerformance Index (PI) a greater than 1. PI is the ratio of mutantresidual activity to parent (i.e., S242Q) residual activity. Results areshown in Tables 18-1 and 18-2.

TABLE 18-1 S242Q CCL - CS-28 rice starch microswatch winners, Tide 2x(North American conditions as described in Ex. 12) Variant # 97 319 358443 rel charge PI 13 Q97E Q319R Q358R Q443E 0 1.44 14 Q97E Q319R Q358RQ443R 2 1.32 15 Q97E Q319R Q358R 1 1.40 16 Q97E Q319R Q443E −1 1.33 17Q97E Q319R Q443R 1 1.40 18 Q97E Q319R 0 1.41 20 Q97E Q358E Q443R −1 1.1523 Q97E Q358R Q443R 1 1.21 25 Q97E Q443E −2 1.18 26 Q97E Q443R 0 1.25 27Q97E −1 1.16 28 Q97R Q319E Q358E Q443E −2 2.32 29 Q97R Q319E Q358E Q443R0 2.54 30 Q97R Q319E Q358E −1 2.93 31 Q97R Q319E Q358R Q443E 0 2.27 32Q97R Q319E Q358R Q443R 2 2.28 33 Q97R Q319E Q358R 1 2.34 34 Q97R Q319EQ443E −1 2.31 35 Q97R Q319E Q443R 1 2.31 36 Q97R Q319E 0 2.14 37 Q97RQ319R Q358E Q443E 0 1.93 38 Q97R Q319R Q358E Q443R 2 1.85 39 Q97R Q319RQ358E 1 2.14 40 Q97R Q319R Q358R Q443E 2 1.92 41 Q97R Q319R Q358R Q443R4 1.37 42 Q97R Q319R Q358R 3 1.61 43 Q97R Q319R Q443E 1 1.90 44 Q97RQ319R Q443R 3 1.64 45 Q97R Q319R 2 1.99 46 Q97R Q358E Q443E −1 1.40 47Q97R Q358E Q443R 1 1.29 48 Q97R Q358E 0 1.60 49 Q97R Q358R Q443E 1 1.5750 Q97R Q358R Q443R 3 1.38 51 Q97R Q358R 2 1.37 52 Q97R Q443E 0 1.51 54Q97R 1 1.51 55 Q319E Q358E Q443E −3 1.14 56 Q319E Q358E Q443R −1 1.38 57Q319E Q358E −2 1.10 58 Q319E Q358R Q443E −1 1.25 59 Q319E Q358R Q443R 11.41 60 Q319E Q358R 0 1.49 61 Q319E Q443E −2 1.16 62 Q319E Q443R 0 1.4563 Q319E −1 1.28 64 Q319R Q358E Q443E −1 1.12 65 Q319R Q358E Q443R 11.19 66 Q319R Q358E 0 1.36 67 Q319R Q358R Q443E 1 1.24 69 Q319R Q358R 21.19 70 Q319R Q443E 0 1.29 76 Q358R Q443E 0 1.22 78 Q358R 1 1.25 79Q443E −1 1.24 80 Q443R 1 1.17

TABLE 18-2 S242Q CCL - CS-28 rice starch microswatch winners, Persil(Western European conditions) Variant # 97 319 358 443 rel charge PI 2Q97E Q319E Q358E Q443R −2 1.41 3 Q97E Q319E Q358E −3 1.94 4 Q97E Q319EQ358R Q443E −2 1.61 5 Q97E Q319E Q358R Q443R 0 1.39 6 Q97E Q319E Q358R−1 2.04 7 Q97E Q319E Q443E −3 2.05 8 Q97E Q319E Q443R −1 1.84 9 Q97EQ319E −2 2.27 10 Q97E Q319R Q358E Q443E −2 1.35 13 Q97E Q319R Q358RQ443E 0 1.45 14 Q97E Q319R Q358R Q443R 2 1.17 15 Q97E Q319R Q358R 1 1.2216 Q97E Q319R Q443E −1 1.26 17 Q97E Q319R Q443R 1 1.29 18 Q97E Q319R 01.76 26 Q97E Q443R 0 1.36 27 Q97E −1 1.31 28 Q97R Q319E Q358E Q443E −22.21 29 Q97R Q319E Q358E Q443R 0 1.96 30 Q97R Q319E Q358E −1 1.94 31Q97R Q319E Q358R Q443E 0 2.11 32 Q97R Q319E Q358R Q443R 2 1.87 33 Q97RQ319E Q358R 1 2.41 34 Q97R Q319E Q443E −1 2.20 35 Q97R Q319E Q443R 12.21 36 Q97R Q319E 0 2.07 37 Q97R Q319R Q358E Q443E 0 1.86 38 Q97R Q319RQ358E Q443R 2 1.83 39 Q97R Q319R Q358E 1 1.99 40 Q97R Q319R Q358R Q443E2 1.85 41 Q97R Q319R Q358R Q443R 4 1.36 42 Q97R Q319R Q358R 3 1.90 43Q97R Q319R Q443E 1 1.99 44 Q97R Q319R Q443R 3 1.94 45 Q97R Q319R 2 1.7546 Q97R Q358E Q443E −1 1.71 47 Q97R Q358E Q443R 1 1.39 48 Q97R Q358E 01.85 50 Q97R Q358R Q443R 3 1.24 51 Q97R Q358R 2 1.36 52 Q97R Q443E 01.25 54 Q97R 1 1.88 55 Q319E Q358E Q443E −3 1.12 56 Q319E Q358E Q443R −11.17 58 Q319E Q358R Q443E −1 1.16 59 Q319E Q358R Q443R 1 1.25 60 Q319EQ358R 0 1.50 63 Q319E −1 1.36 64 Q319R Q358E Q443E −1 1.10 65 Q319RQ358E Q443R 1 1.18 66 Q319R Q358E 0 1.25 67 Q319R Q358R Q443E 1 1.29 70Q319R Q443E 0 1.15

Activity was also measured using the BODIPY starch hydrolysis assay asprovided herein. The results are shown in Table 18-3. The relativespecific activity on this starch substrate (a corn starch) greater than1 indicates the variant has higher specific activity than the S242Qparent. Relative ppm is expression titers, greater than 1 indicateshigher titers (in shake tubes) than the S242Q parent.

TABLE 18-3 S242Q CCL - titer and/or BODIPY-starch winners Rel Rel SpVariant # 97 319 358 443 Charge ppm act 1 Q97E Q319E Q358E Q443E −4 1.271.29 2 Q97E Q319E Q358E Q443R −2 1.19 1.31 3 Q97E Q319E Q358E −3 1.001.43 4 Q97E Q319E Q358R Q443E −2 1.23 1.43 5 Q97E Q319E Q358R Q443R 00.94 1.78 6 Q97E Q319E Q358R −1 0.89 1.81 7 Q97E Q319E Q443E −3 1.401.41 8 Q97E Q319E Q443R −1 1.12 1.58 9 Q97E Q319E −2 1.09 1.56 10 Q97EQ319R Q358E Q443E −2 1.45 1.32 11 Q97E Q319R Q358E Q443R 0 1.32 1.49 12Q97E Q319R Q358E −1 1.58 1.27 13 Q97E Q319R Q358R Q443E 0 0.65 1.44 14Q97E Q319R Q358R Q443R 2 0.66 1.65 15 Q97E Q319R Q358R 1 0.80 1.64 16Q97E Q319R Q443E −1 1.09 1.51 17 Q97E Q319R Q443R 1 1.00 1.42 18 Q97EQ319R 0 0.87 1.78 19 Q97E Q358E Q443E −3 1.22 0.88 21 Q97E Q358E −2 1.120.88 22 Q97E Q358R Q443E −1 0.91 1.16 23 Q97E Q358R Q443R 1 0.78 1.25 24Q97E Q358R 0 1.08 1.14 25 Q97E Q443E −2 1.12 1.00 28 Q97R Q319E Q358EQ443E −2 0.78 1.87 29 Q97R Q319E Q358E Q443R 0 0.80 1.81 30 Q97R Q319EQ358E −1 0.68 2.21 31 Q97R Q319E Q358R Q443E 0 0.68 1.96 32 Q97R Q319EQ358R Q443R 2 0.70 2.05 33 Q97R Q319E Q358R 1 0.60 2.27 34 Q97R Q319EQ443E −1 0.65 2.25 35 Q97R Q319E Q443R 1 0.70 2.15 36 Q97R Q319E 0 0.732.23 37 Q97R Q319R Q358E Q443E 0 0.93 2.11 38 Q97R Q319R Q358E Q443R 20.65 2.21 39 Q97R Q319R Q358E 1 0.82 2.22 40 Q97R Q319R Q358R Q443E 20.74 2.28 41 Q97R Q319R Q358R Q443R 4 0.55 2.09 42 Q97R Q319R Q358R 30.67 2.48 43 Q97R Q319R Q443E 1 0.84 2.35 44 Q97R Q319R Q443R 3 0.732.41 45 Q97R Q319R 2 0.76 2.45 46 Q97R Q358E Q443E −1 0.79 1.45 47 Q97RQ358E Q443R 1 0.75 1.42 48 Q97R Q358E 0 0.82 1.46 49 Q97R Q358R Q443E 10.67 1.69 50 Q97R Q358R Q443R 3 0.60 1.60 51 Q97R Q358R 2 0.64 1.29 52Q97R Q443E 0 0.83 1.43 54 Q97R 1 0.72 1.49 55 Q319E Q358E Q443E −3 0.991.15 56 Q319E Q358E Q443R −1 0.77 1.40 57 Q319E Q358E −2 0.83 1.34 58Q319E Q358R Q443E −1 0.73 1.49 59 Q319E Q358R Q443R 1 0.67 1.61 60 Q319EQ358R 0 0.80 1.67 61 Q319E Q443E −2 0.91 1.39 62 Q319E Q443R 0 0.73 1.4563 Q319E −1 0.75 1.41 64 Q319R Q358E Q443E −1 1.05 1.28 65 Q319R Q358EQ443R 1 0.94 1.42 66 Q319R Q358E 0 0.96 1.39 67 Q319R Q358R Q443E 1 1.021.50 68 Q319R Q358R Q443R 3 0.71 1.57 69 Q319R Q358R 2 0.71 1.58 70Q319R Q443E 0 0.91 1.49 72 Q319R 1 0.95 1.56 77 Q358R Q443R 2 0.67 1.2278 Q358R 1 0.66 1.15

Example 19 Balancing Mutational Effects on Amylase Activity andExpression

This example illustrates that two separate enzyme properties can besimultaneously optimized by the introduction of multiple amino acidsubstitutions, even where the properties are negatively correlated due,for example, to oppositely linked to charge characteristics of theprotein.

It was determined during experimentation that the median expression ofAmyS-242Q decreased with increasing positive charge. However, specificBODIPY starch hydrolysis increased with increasing positive charge.Enhanced recombinant amylase expression and starch hydrolysis aredesirable in an engineered variant of AmyS-242Q suitable for starchliquefaction in the fuel ethanol industry or cleaning in detergentapplications for instance. These properties, however, are apparentlyconflicting properties. Using the methods provided herein, it ispossible to produce a more highly expressed amylase variant withoutseverely compromising starch hydrolysis by selectively combining singlemutations. The strategy described herein was successfully used toproduce and select multiply-substituted AmyS-242Q variants havingimprovements in a first property (e.g., expression as the primaryproperty), while improving or not sacrificing a second property (e.g.,starch hydrolysis as the secondary property).

In addition, in converse to median expression of AmyS-242Q variants,cornstarch microswatch cleaning increased with increasing positivecharge. Enhanced recombinant amylase expression and cleaning performanceare desirable in an engineered variant of AmyS-242Q. These properties,however, are also apparently conflicting properties. Using the methodsdisclosed herein, it is possible to produce a more highly expressedamylase variant without severely compromising cleaning performance byselectively combining single mutations. The strategy described hereinwas successfully used to produce and select multiply-substitutedAmyS-242Q variants having improvements in a first property (e.g.,expression as the primary property), while improving or not sacrificinga second property (e.g., rice starch microswatch cleaning as thesecondary property).

In particular, an eighty member AmyS-S242Q charge combinatorial library(CCL) comprising variants having combinations of from one to foursubstitutions of charged residues was tested for shake tube expression,BODIPY-starch hydrolysis, and rice starch cleaning activity. AmyS-S242Qwinners are shown in Tables 7-1 and 7-1. Importantly, themultiply-substituted variants of Table 19-1 have equal or improvedexpression and equal or improved BODIPY-starch hydrolysis as compared tothe parent enzyme. Similarly, the multiply-substituted variants of Table19-2 have equal or improved expression and equal or improved rice starchcleaning activity as compared to the parent enzyme.

TABLE 19-1 AmyS-S242Q Expression and BODIPY-Starch Hydrolysis WinnersEx- pres- sion BODIPY Variant 97 319 358 443 Charge (PI) (PI) 1 Q97EQ319E Q358E Q443E −4 1.27 1.29 2 Q97E Q319E Q358E Q443R −2 1.19 1.31 3Q97E Q319E Q358E −3 1.00 1.43 4 Q97E Q319E Q358R Q443E −2 1.23 1.43 7Q97E Q319E Q443E −3 1.40 1.41 8 Q97E Q319E Q443R −1 1.12 1.58 9 Q97EQ319E −2 1.09 1.56 10 Q97E Q319R Q358E Q443E −2 1.45 1.32 11 Q97E Q319RQ358E Q443R 0 1.32 1.49 12 Q97E Q319R Q358E −1 1.58 1.27 16 Q97E Q319RQ443E −1 1.09 1.51 17 Q97E Q319R Q443R +1 1.00 1.42 24 Q97E Q358R 0 1.081.14 25 Q97E Q443E −2 1.12 1.00 64 Q319R Q358E Q443E −1 1.05 1.28 67Q319R Q358R Q443E +1 1.02 1.50

TABLE 19-2 AmyS-S242Q Expression and Rice-Starch Hydrolysis WinnersExpres- Variant 97 319 358 443 Charge sion CS-28 1 Q97E Q319E Q358EQ443E −4 1.27 1.01 11 Q97E Q319R Q358E Q443R 0 1.32 1.18 12 Q97E Q319RQ358E −1 1.58 1.13 16 Q97E Q319R Q443E −1 1.09 1.43 17 Q97E Q319R Q443R+1 1.00 1.55 24 Q97E Q358R 0 1.08 1.15 25 Q97E Q443E −2 1.12 1.09 64Q319R Q358E Q443E −1 1.05 1.18 67 Q319R Q358R Q443E +1 1.02 1.15

In sum, because enzyme activity and enzyme production have differentcharge dependencies (see FIGS. 26A, 26B, 27A and 27B) they arenegatively correlated (see FIGS. 25A and 25B). However, there are anumber of variants that are improved in both expression and activity,and analyzing the library in this manner allows them to be identified.

Although demonstrated with amylases this method is applicable to otherenzyme classes such as proteases, lipases, cellulases, transferases andpectinases. Moreover any combination of two or more properties can beanalyzed simultaneously such as expression, activity, binding, thermalstability, detergent and chelant stability.

Example 20 Further Characterization of S242Variants

A library of S242 variants (S242A, 242A, 242C, 242D, 242E, 242F, 242G,242H, 242I, 242K, 242L, 242M, 242N, 242P, 242Q, 242R, 242T, 242V, 242W,and 242Y) were further characterized to determine the ProteinExpression, Specific Activity at pH 5.8 and pH 4, Specific Activity onCorn Flour 85, and % Residual Activity at Temperature (see also Example3). Results are shown with relative comparison to wild-type (or S242Sparent amylase) and the control enzyme (SPEZYME ETHYL).

Data are shown in Table 20-1.

The specific activity at pH 5.8 and pH 4 was measured using theAlpha-Amylase Activity on Maltoheptaose Assay (pH StabilityDetermination) as follows:

The alpha-amylase activity of B. subtilis AmyS and AmyS variants onmaltoheptaose at pH 5.8 and pH 4 was measured by monitoring productionof glucose, using an enzyme-coupled colorimetric kinetic assay. Enzymereactions were carried out in flat-bottom polystyrene 96-well microtiterplates at room temperature. For the assay conducted at pH 5.8, 10 μL ofculture supernatant of AmyS and AmyS variants were mixed with 40 μL ofbuffer containing sodium acetate (pH 5.8), CaCl₂, Tween-20, horseradishperoxidase (Sigma-Aldrich, cat. No. 8375) and glucose oxidase (GenencorOxyGo™), at concentrations such that the final 50 μL volume contained 50mM, 2.6 mM, 0.005% (w/v), 20 U/ml and 50 U/ml of each component,respectively. Reactions were initiated by the addition of 50 μl ofbuffer containing 50 mM sodium acetate (pH 5.8), 5.4 mg/mL2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt(Sigma-Aldrich, cat. no. A1888) and 10 mM maltoheptaose (Sigma-Aldrich,cat. No. M7753), and was followed by 5 seconds of mixing. Colorformation in the reaction was monitored at 405 nm in 9 second intervalsfor 240 seconds using a SpectraMAX 250 spectrophotometer (MolecularDevices). Enzyme activity was reported as the rate of color formationduring the 120-240 second interval of monitoring. For the assayconducted at pH 4.0, the method as described above was repeated exactlyexcept using buffers at pH 4.0.

TABLE 20-1 Characterization of S242 Variants Specific Specific SpecificResidual Protein Activity Activity Activity Activity Variant ExpressionpH 5.8 pH 4 Corn Flour 85 (%) 242A 133 0.189 0.0540 0.0112 59.5 242C 950.193 0.0502 0.0109 29.3 242D 176 0.140 0.0344 0.0045 41.6 242E 1790.151 0.0400 0.0145 44.6 242F 116 0.162 0.0441 0.0072 33.8 242G 1720.164 0.0444 0.0102 31.6 242H 174 0.145 0.0414 0.0074 19.3 242I 1150.154 0.0445 0.0118 27.0 242K 187 0.148 0.0444 0.0083 22.8 242L 1200.202 0.0729 0.0114 28.5 242M 95 0.241 0.0735 0.0122 42.9 242N 170 0.1610.0419 0.0082 30.2 242P 168 0.149 0.0322 0.0041 6.7 242Q 142 0.1520.0374 0.0177 61.3 242R 176 0.154 0.0368 0.0062 13.0 wildtype 135 0.1640.0367 0.0122 34.0 242T 165 0.145 0.0334 0.0081 24.8 242V 106 0.1680.0407 0.0089 22.3 242W 112 0.199 0.0471 0.0083 5.6 242Y 127 0.1890.0541 0.0077 15.7 pos 75 0.189 0.0445 0.0311 85.0 wildtype 156 0.1530.0416 0.0068 32.3

All publications and patents mentioned in the above specification areincorporated herein by reference. Although the disclosed methods andenzymes have in some instances been described in connection withspecific or preferred embodiments, it should be understood what iscovered by the appended claims is not limited to such specific orpreferred embodiments. Indeed, various modifications and variations ofthe disclosed methods and enzymes will be apparent to those skilled inthe art, and various modifications of the described modes for practicingwhat has been disclosed are included within the scope of the followingclaims.

1. An alpha-amylase variant comprising an amino acid sequence at least95% identical to a wild-type alpha-amylase polypeptide sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 16, and having asubstitution at an amino acid position corresponding to position 242 ofSEQ ID NO: 1, wherein said variant has alpha-amylase activity and aT_(m) about 5-10° C. higher than said wild-type alpha-amylase; andwherein the substitution at an amino acid position corresponding toposition 242 of SEQ ID NO: 1 is selected from the group consisting ofS242A, S242E, and S242Q.
 2. The alpha-amylase variant of claim 1,wherein the amino acid sequence is at least 98% identical to theAmyS-like alpha-amylase polypeptide sequence of SEQ ID NO:
 1. 3. Anisolated polynucleotide encoding an alpha-amylase variant comprising anamino acid sequence at least 95% identical to a wild-type alpha-amylasepolypeptide sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, and SEQ IDNO: 16, and having a substitution at an amino acid positioncorresponding to position 242 of SEQ ID NO: 1, wherein said variant hasalpha-amylase activity and a T_(m) about 5-10° C. higher than saidwild-type alpha-amylase; and wherein the substitution at an amino acidposition corresponding to position 242 of SEQ ID NO: 1 is selected fromthe group consisting of S242A, S242E, and S242Q.
 4. A vector comprisingthe isolated polynucleotide of claim
 3. 5. An isolated host cellcomprising the isolated polynucleotide of claim 2 or the vector of claim4.
 6. The isolated host cell of claim 5 that is a Bacillus subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, B. thuringiensis, Streptomyces lividans, S. murinus; Escherichiacoli, or a Pseudomonas spp.
 7. A composition comprising: a) thealpha-amylase variant of claim 1, and b) at least one additional enzyme.8. The composition of claim 7, wherein the additional enzyme is aphytase.
 9. The composition of claim 8, wherein the alpha-amylasevariant and the phytase are present in amounts such that the ratio ofalpha-amylase units to phytase units (AAU:FTU) is about 1:15 to about15:1.
 10. The composition of claim 8, wherein the alpha-amylase variantand the phytase are present in amounts such that the ratio of AAU:FTU isabout 1:4 to about 3:1.
 11. The composition of claim 8, wherein thephytase has a sequence that is SEQ ID NO:
 31. 12. A method of treating astarch slurry comprising: a) adding to the starch slurry at least onephytase and at least one alpha-amylase variant of claim 1; wherein thephytase and the alpha-amylase variant are added at or about the sametime, or separately in any order; and b) incubating the starch slurryunder conditions permissive of activity of the phytase and thealpha-amylase variant.
 13. The method of claim 12, wherein the phytaseis added before the alpha-amylase variant.
 14. The method of claim 12,wherein the starch slurry is preincubated after adding the phytase andbefore adding the alpha-amylase variant.
 15. The method of claim 12,wherein the inclusion of the phytase results in an increase inthermostability of the alpha-amylase variant relative to a comparablemethod that does not include contacting the starch slurry with phytase.16. The method of claim 12, wherein the phytase and the alpha-amylasevariant are present in a single blend before adding to the starchslurry.
 17. The method of claim 12, wherein the phytase has the aminoacid sequence of SEQ ID NO:
 31. 18. A method of producing a fermentablesubstrate from a starch-containing slurry comprising milled grain, themethod comprising: a) contacting the starch-containing slurry with atleast one phytase and at least one alpha-amylase variant of claim 1 inan amount sufficient to produce a fermentable substrate from thestarch-containing slurry; wherein the contact with the phytase and thealpha-amylase variant is initiated at or about the same time, orseparately in any order; and b) incubating the starch-containing slurryunder conditions permissive of activity of the phytase and thealpha-amylase variant for a time that allows production of thefermentable substrate; wherein when the contact with the phytase isinitiated before the alpha-amylase variant, the starch-containing slurryis incubated at a temperature that is 0-30° C. less than agelatinization temperature prior to contacting the starch-containingslurry with the alpha-amylase variant, after which the temperature israised above the gelatinization temperature for a time effective tohydrolyze starch.
 19. A method of treating a starch-containing materialor a starch comprising contacting the starch-containing material or thestarch with a composition comprising at least one alpha-amylase variantof claim 1 under conditions sufficient to allow detectable activity ofthe alpha-amylase variant, wherein the starch-containing material or thestarch is at least partially degraded by the alpha-amylase variant. 20.The method of claim 19 further comprising at least one additional enzymewhich is a phytase, cellulase, protease, aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, chitinase, cutinase,cyclodextrin glucanotransferase, deoxyribonuclease, esterase,α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase,β-glucosidase, haloperoxidase, invertase, isomerase, laccase, lipase,mannosidase, oxidase, pectinase, peptidoglutaminase, peroxidase,polyphenoloxidase, nuclease, ribonuclease, transglutaminase, xylanase,pullulanase, isoamylase, carrageenase, or a combination of two or moreof the foregoing.
 21. The method of claim 19 that is part of a processfor starch degradation, liquefaction, fermentation, alcohol production,sweetener production, production of a fermentable substrate, cleaning,washing, stain removal, or baking process.
 22. A kit comprising, in oneor more packages provided as a unit: i) at least one alpha-amylasevariant of claim 1; and ii) at least one additional enzyme.
 23. The kitof claim 22 further comprising instructions for using the alpha-amylasevariant and the at least one additional enzyme in a useful processinvolving enzymatic cleavage of starch molecules.
 24. The kit of claim22, wherein the additional enzyme is a phytase.
 25. The alpha-amylasevariant of claim 1, further comprising an amino acid substitution at anamino acid position corresponding to position 97, 319, 358, or 443 ofSEQ ID NO:
 1. 26. The alpha-amylase variant of claim 1, wherein thevariant comprises an amino acid sequence at least 95% identical to thealpha-amylase polypeptide sequence selected from the group consisting ofSEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:11.
 27. The alpha-amylase variant of claim 1, wherein the variantcomprises an amino acid sequence at least 95% identical to thealpha-amylase polypeptide sequence selected from the group consisting ofSEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:
 11. 28. Thealpha-amylase variant of claim 1, wherein the variant comprises an aminoacid sequence at least 95% identical to the alpha-amylase polypeptidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 16.